LSR receptor, its activity, its cloning, and its applications to the diagnosis, prevention and/or treatment of obesity and related risks or complications

ABSTRACT

The present invention relates to a new complex receptor polypeptide LSR (Lipolysis Stimulated Receptor), characterized by its functional activities, the cloning of the cDNAs complementary to the messenger RNAs encoding each of the subunits of the multimeric complex, vectors and transformed cells, methods of diagnosis and of selection of compounds which can be used as medicament for the prevention and/or treatment of pathologies and/or of pathogeneses such as obesity and anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, and more generally the various pathologies associated with abnormalities in the metabolism of cytokines.

INTRODUCTION

[0001] The present invention relates to a new complex receptorpolypeptide LSR (Lipolysis Stimulated Receptor), characterized by itsfunctional activities, the cloning of the cDNAs complementary to themessenger RNAs encoding each of the subunits of the multimeric complex,vectors and transformed cells, methods of diagnosis and of selection ofcompounds which can be used as medicament for the prevention and/ortreatment of pathologies and/or of pathogeneses such as obesity andanorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, andmore generally the various pathologies associated with abnormalities inthe metabolism of cytokines.

[0002] Obesity is a public health problem which is both serious andwidespread: in industrialized countries, a third of the population hasan excess weight of at least 20% relative to the ideal weight. Thephenomenon continues to worsen, in regions of the globe whose economiesare being modernized, such as the Pacific islands, and in general. Inthe United States, the number of obese people has passed from 25% at theend of the 70s to 33% at the beginning of the 90s.

[0003] Obesity considerably increases the risk of developingcardiovascular or metabolic diseases. It is estimated that if the entirepopulation had an ideal weight, the risk of coronary insufficiency woulddecrease by 25% and that of cardiac insufficiency and of cerebralvascular accidents by 35%. Coronary insufficiency, atheromatous diseaseand cardiac insufficiency are at the forefront of the cardiovascularcomplications induced by obesity. For an excess weight greater than 30%,the incidence of coronary diseases is doubled in subjects under 50years. Studies carried out for other diseases are equally eloquent. Foran excess weight of 20%, the risk of high blood pressure is doubled. Foran excess weight of 30%, the risk of developing a non-insulin-dependentdiabetes is tripled. That of hyperlipidemias is multiplied by 6.

[0004] The list of diseases whose onset is promoted by obesity is long:hyperuricemia (11.4% in obese subjects, against 3.4% in the generalpopulation), digestive pathologies, abnormalities in hepatic functions,and even certain cancers.

[0005] Whether the physiological changes in obesity are characterized byan increase in the number of adipose cells, or by an increase in thequantity of triglycerides stored in each adipose cell, or by both, thisexcess weight results mainly from an imbalance between the quantities ofcalories consumed and those of the calories used by the body. Studies onthe causes of this imbalance have been in several directions. Some havefocused on studying the mechanism of absorption of foods, and thereforethe molecules which control food intake and the feeling of satiety.Other studies have been related to the basal metabolism, that is to saythe manner in which the body uses the calories consumed.

[0006] The treatments for obesity which have been proposed are of fourtypes. Food restriction is the most frequently used. The obeseindividuals are advised to change their dietary habits so as to consumefewer calories. This type of treatment is effective in the short-term.However, the recidivation rate is very high. The increase in calorie usethrough physical exercise is also proposed. This treatment isineffective when applied alone, but it improves, however, weight loss insubjects on a low-calorie diet. Gastrointestinal surgery, which reducesthe absorption of the calories ingested, is effective but has beenvirtually abandoned because of the side effects which it causes. Themedicinal approach uses either the anorexigenic action of moleculesinvolved at the level of the central nervous system, or the effect ofmolecules which increase energy use by increasing the production ofheat. The prototypes of this type of molecule are the thyroid hormoneswhich uncouple oxidative phosphorylations of the mitochondrialrespiratory chain. The side effects and the toxicity of this type oftreatment make their use dangerous. An approach which aims to reduce theabsorption of dietary lipids by sequestering them in the lumen of thedigestive tube is also in place. However, it induces physiologicalimbalances which are difficult to tolerate: deficiency in the absorptionof fat-soluble vitamins, flatulence and steatorrhoea. Whatever theenvisaged therapeutic approach, the treatments of obesity are allcharacterized by an extremely high recidivation rate.

[0007] The molecular mechanisms responsible for obesity in humans arecomplex and involve genetic and environmental factors. Because of thelow efficiency of the treatments known up until now, it is urgent todefine the genetic mechanisms which determine obesity, so as to be ableto develop better targeted medicaments.

[0008] More than 20 genes have been studied as possible candidates,either because they have been implicated in diseases of which obesity isone of the clinical manifestations, or because they are homologues ofgenes involved in obesity in animal models. Situated in the 7q31chromosomal region, the OB gene is one of the most widely studied. Itsproduct, leptin, is involved in the mechanisms of satiety. Leptin is aplasma protein of 16 kDa produced by the adipocytes under the action ofvarious stimuli. Obese mice of the ob/ob type exhibit a deficiency inthe leptin gene; this protein is undetectable in the plasma of theseanimals. The administration of leptin obtained by genetic engineering toob/ob mice corrects their relative hyperphagia and allows normalizationof their weight. This anorexigenic effect of leptin calls into play areceptor of the central nervous system: the ob receptor which belongs tothe family of class 1 cytokine receptors. The ob receptor is deficientin obese mice of the db/db strain. The administration of leptin to thesemice has no effect on their food intake and does not allow substantialreduction in their weight. The mechanisms by which the ob receptorstransmit the signal for satiety are not precisely known. It is possiblethat neuropeptide Y is involved in this signalling pathway. It isimportant to specify at this stage that the ob receptors are not theonly regulators of appetite. The Melanocortin 4 receptor is alsoinvolved since mice made deficient in this receptor are obese (Gura,1997).

[0009] The discovery of leptin and the characterization of the leptinreceptor at the level of the central nervous system have opened a newroute for the search for medicaments against obesity. This model,however, rapidly proved disappointing. Indeed, with only one exception(Montague et al., 1997), the genes encoding leptin or its ob receptorhave proved to be normal in obese human subjects. Furthermore andparadoxically, the plasma concentrations of leptin, the satiety hormone,are abnormally high in most obese human subjects. Most of thetherapeutic research efforts in this direction have centred on thecharacterization of the effect of leptin at the level of the centralnervous system.

SUMMARY OF THE INVENTION

[0010] The present invention results from a focusing of the researcheffort on the discovery of the mechanisms of leptin elimination. Themost widely accepted working hypothesis is that the plasma levels ofleptin are high in obese subjects because this hormone is produced bythe adipose tissue and that the fatty mass is increased in obesesubjects. The inventors have formulated a different hypothesis and havepostulated that the concentrations of leptin are increased in obeseindividuals because the clearance of this hormone is reduced. Thisdeficiency causes a leptin resistance syndrome and the obese individualdevelops a suitable response to the high concentrations of leptin. Inthis perspective, the treatment of obese subjects ought to consist notin an increase in the leptin levels but in a normalization thereof. Atthis stage, it is essential to recall that the ob type receptors aresignalling type receptors. These receptors can bind leptin at the levelof the plasma membrane but cannot cause the protein to enter inside thecell for it to be degraded therein. The ob receptors are not endocytosisreceptors.

[0011] LSR Receptor

[0012] The inventors have characterized a receptor, in particularhepatic, called LSR receptor, whose activity is dual. The LSR receptorallows, on the one hand, endocytosis of lipoproteins, when it isactivated by the free fatty acids, thus serving as a pathway for theclearance of lipoproteins. This pathway serves mainly, but notexclusively, for the clearance of particles high in triglycerides ofintestinal origin (Mann et al., 1995). This activity, expressed mostparticularly at the hepatic level, is dependent on the presence of freefatty acids which, by binding to the receptor, induce a reversiblechange in the conformation of this complex and allow it to bind, with ahigh affinity, various classes of lipoproteins such as those containingapoprotein B or apoprotein E.

[0013] On the other hand, under normal conditions, in the absence offree fatty acids, the complex receptor LSR does not bind lipoproteins,but is capable of binding a cytokine, in particular leptin, and then ofinternalizing it and of degrading it.

[0014] The present invention therefore relates to a purified LSRreceptor, in particular of hepatic cells, characterized in that it iscapable, in the presence of free fatty acids, of binding lipoproteins,and in the absence of free fatty acids, of binding a cytokine,preferably leptin.

[0015] According to the invention, this LSR receptor is, in addition,characterized in that the bound lipoproteins or the bound cytokine areincorporated into the cell and then degraded, the bound lipoproteinscontaining in particular apoprotein B or E.

[0016] It should be understood that the invention does not relate to theLSR receptors in a natural form, that is to say that they are not takenin their natural environment but obtained by purification from naturalsources, or alternatively obtained by genetic recombination, oralternatively by chemical synthesis and capable, in this case, ofcontaining non-natural amino acids, as will be described below. Theproduction of a recombinant LSR receptor, which may be carried out usingone of the nucleotide sequences according to the invention, isparticularly advantageous because it makes it possible to obtain anincreased level of purity of the receptor.

[0017] More particularly, the invention relates to a purified rat LSRreceptor, characterized in that it comprises at least one subunit havinga molecular weight of about 66 kDa and a subunit having a molecularweight of about 58 kDa.

[0018] Preferably, the purified rat LSR receptor of the presentinvention is characterized in that it contains an a subunit comprisingthe amino acid sequence of SEQ ID 2 or a sequence homologous thereto, oran α′ subunit comprising the amino acid sequence of SEQ ID 4 or asequence homologous thereto, and one, preferably three, β subunitscomprising the amino acid sequence of SEQ ID 6 or a sequence homologousthereto.

[0019] The invention also relates to a purified mouse LSR receptor,characterized in that it comprises at least one subunit having amolecular weight of about 66 kDa and a subunit having a molecular weightof about 58 kDa.

[0020] Preferably, the purified mouse LSR receptor of the presentinvention is characterized in that it contains an a subunit comprisingthe amino acid sequence of SEQ ID 16 or a sequence homologous thereto,or an α′ subunit comprising the amino acid sequence of SEQ ID 17 or asequence homologous thereto, and one, preferably three, β subunitscomprising the amino acid sequence of SEQ ID 18 or a sequence homologousthereto.

[0021] The invention also relates to a purified human LSR receptor,characterized in that it comprises at least one subunit having amolecular weight of about 72 kDa and a subunit having a molecular weightof about 64 kDa.

[0022] Preferably, the purified human LSR receptor of the presentinvention is characterized in that it contains an α′ subunit comprisingthe amino acid sequence of SEQ ID 8 or a sequence homologous thereto, oran α′ subunit comprising the amino acid sequence of SEQ ID 10 or asequence homologous thereto, and one, preferably three, β subunitscomprising the amino acid sequence of SEQ ID 12 or a sequence homologousthereto.

[0023] A particularly preferred embodiment of the LSR receptors of thepresent invention is a recombinant LSR receptor obtained by expressing,in a recombinant host, one or more nucleotide sequences according to theinvention. This preferred recombinant receptor consists of an α or α′subunit and one, preferably three, β subunits, in particular an a or α′subunit and three p subunits of a human LSR receptor.

[0024] Polypeptide sequences of LSR

[0025] The invention relates to polypeptides, characterized in that theyare a constituent of an LSR receptor according to the invention.

[0026] It should be understood that the invention does not relate to thepolypeptides in a natural form, that is to say that they are not takenin their natural environment. Indeed, the invention relates to thepeptides obtained by purification from natural sources, or alternativelyobtained by genetic recombination, or alternatively by chemicalsynthesis, and capable, in this case, of containing non-natural aminoacids, as will be described below. The production of a recombinantpolypeptide, which may be carried out using one of the nucleotidesequences according to the invention or a fragment of one of thesesequences, is particularly advantageous because it makes it possible toobtain an increased level of purity of the desired polypeptide.

[0027] The invention therefore relates to a purified, isolated orrecombinant polypeptide comprising a sequence of at least 5, preferablyat least 10 to 15, consecutive amino acids of an LSR receptor, as wellas the homologues, equivalents or variants of the said polypeptide, orone of their fragments. Preferably, the sequence of at least 10 to 15amino acids of the LSR receptor is a biologically active fragment of anLSR receptor.

[0028] More particularly, the invention relates to purified, isolated orrecombinant polypeptides comprising a sequence of at least 10 to 15amino acids of a rat LSR receptor, of a mouse LSR receptor or of a humanLSR receptor.

[0029] In the present description, the term polypeptide will be used toalso designate a protein or a peptide.

[0030] Nucleotide sequences of LSR

[0031] The subject of the present invention is also purified nucleicacid sequences, characterized in that they encode an LSR receptor or apolypeptide according to the invention.

[0032] The invention relates to a purified nucleic acid, characterizedin that it comprises at least 8, preferably at least 10 and moreparticularly at least 15 consecutive nucleotides of the polynucleotideof a genomic, cDNA or RNA sequence of the LSR receptor, as well as thenucleic acid sequences complementary to this nucleic acid. Moreparticularly, the invention relates to the purified, isolated orrecombinant nucleic acids comprising a sequence of at least 8,preferably at least 10 and more particularly at least 15 consecutivenucleotides of the polynucleotide of a nucleic sequence of a mouse LSRreceptor or of a human LSR receptor.

[0033] The invention also relates to the variant, mutated, equivalent orhomologous nucleic sequences of the nucleic sequences according to theinvention, or one of their fragments. It finally relates to thesequences capable of hybridizing specifically with the nucleic sequencesaccording to the invention.

[0034] The invention therefore also relates to the nucleic acidsequences contained in the gene encoding the LSR receptor, in particulareach of the exons of the said gene or a combination of exons of the saidgene, or alternatively a polynucleotide extending over a portion of oneor more exons. Preferably, these nucleic acids encode one or morebiologically active fragments of the human LSR receptor.

[0035] The present invention also relates to the purified nucleic acidsequences encoding one or more elements for regulating the expression ofthe LSR gene. Also included in the invention are the nucleic acidsequences of the promoter and/or regulator of the gene encoding thereceptor according to the invention, or one of their allelic variants,the mutated, equivalent or homologous sequences, or one of theirfragments.

[0036] The invention also relates to the purified nucleic sequences forhybridization comprising at least 8 nucleotides, characterized in thatthey can hybridize specifically with a nucleic sequence according to theinvention.

[0037] Preferably, nucleic acid fragments or oligonucleotides, having assequences the nucleotide sequences according to the invention can beused as probes or primers.

[0038] The invention also comprises methods for screening cDNA andgenomic DNA libraries, for the cloning of the isolated cDNAs and/or thegenes coding for the receptor according to the invention, and for theirpromoters and/or regulators, characterized in that they use a nucleicsequence according to the invention.

[0039] The nucleic sequences, characterized in that they are capable ofbeing obtained by one of the preceding methods according to theinvention or the sequences capable of hybridizing with the saidsequences, form part of the invention.

[0040] Vectors, Host Cells and Transgenic Animals

[0041] The invention also comprises the cloning and/or expressionvectors containing a nucleic acid sequence according to the invention.

[0042] The vectors according to the invention, characterized in thatthey comprise elements allowing the expression and/or the secretion ofthe said sequences in a host cell, also form part of the invention.

[0043] The invention comprises, in addition, the host cells, inparticular the eukaryotic and prokaryotic cells, transformed with thevectors according to the invention, as well as the mammals, except man,comprising one of the said transformed cells according to the invention.

[0044] Among the mammals according to the invention, there will bepreferred animals such as mice, rats or rabbits, expressing apolypeptide according to the invention, the phenotype corresponding tothe normal or variant LSR receptor, in particular mutated of humanorigin.

[0045] These cells and animals can be used in a method of producing arecombinant polypeptide according to the invention and can also serve asa model for analysis and screening.

[0046] The invention also relates to the use of a cell, of a mammal orof a polypeptide according to the invention for studying the expressionand the activity of the receptor according to the invention, and thedirect or indirect interactions between the said receptor and chemicalor biochemical compounds which may be involved in the activity of thesaid receptor.

[0047] The invention also relates to the use of a cell, of a mammal orof a polypeptide according to the invention for screening a chemical orbiochemical compound capable of interacting directly or indirectly withthe receptor according to the invention, and/or capable of modulatingthe expression or the activity of the said receptor.

[0048] Production of Polypeptides Derived From the LSR Receptor

[0049] The invention also relates to the synthesis of synthetic orrecombinant polypeptides of the invention, in particular by chemicalsynthesis or using a nucleic acid sequence according to the invention.

[0050] The polypeptides obtained by chemical synthesis and capable ofcomprising non-natural amino acids corresponding to the said recombinantpolypeptides are also included in the invention.

[0051] The method of producing a polypeptide of the invention inrecombinant form is itself included in the present invention, and ischaracterized in that the transformed cells are cultured underconditions allowing the expression of a recombinant polypeptide having apolypeptide sequence according to the invention, and in that the saidrecombinant polypeptide is recovered.

[0052] The recombinant polypeptides, characterized in that they arecapable of being obtained by the said method of production, also formpart of the invention.

[0053] Antibodies

[0054] The mono- or polyclonal antibodies or fragments thereof, chimericor immunoconjugated antibodies, characterized in that they are capableof specifically recognizing a polypeptide or a receptor according to theinvention, form part of the invention.

[0055] There may be noted in particular the advantage of antibodiesspecifically recognizing certain polypeptides, variants or fragments,which are in particular biologically active, according to the invention.

[0056] The invention also relates to methods for the detection and/orpurification of a polypeptide according to the invention, characterizedin that they use an antibody according to the invention.

[0057] The invention comprises, in addition, purified polypeptides,characterized in that they are obtained by a method according to theinvention.

[0058] Moreover, in addition to their use for the purification ofpolypeptides, the antibodies of the invention, in particular themonoclonal antibodies, may also be used for the detection of thesepolypeptides in a biological sample.

[0059] More generally, the antibodies of the invention may beadvantageously used in any situation where the expression, normal orabnormal, of a polypeptide of the LSR receptor, normal or mutated, needsto be observed.

[0060] Detection of Allelic Variability and Diagnosis

[0061] Also forming part of the invention are the methods for thedetermination of an allelic variability, a mutation, a deletion, a lossof heterozygosity or a genetic abnormality, characterized in that theyuse a nucleic acid sequence or an antibody according to the invention.

[0062] These methods relate to, for example, the methods for thediagnosis of the predisposition to obesity, to the associated risks, orto pathologies associated with abnormalities in the metabolism ofcytokines, by determining, in a biological sample from the patient, thepresence of mutations in at least one of the sequences described above.The nucleic acid sequences analysed may be either the genomic DNA, thecDNA or the mRNA.

[0063] Nucleic acids or antibodies based on the present invention canalso be used to allow a positive and differential diagnosis in a patienttaken in isolation, or a pre-symptomatic diagnosis in an at risksubject, in particular with a familial history.

[0064] In addition, the detection of a specific mutation may allow anevolutive diagnosis, in particular as regards the intensity of thepathology or the probable period of its appearance.

[0065] Screening of Compounds of Interest

[0066] Also included in the invention are the methods for selectingchemical or biochemical compounds capable of interacting, directly orindirectly, with the receptor or the polypeptide or nucleotide sequencesaccording to the invention, and/or allowing the expression or theactivity of the LSR receptor to be modulated.

[0067] The invention relates in particular to a method for selectingchemical or biochemical compounds capable of interacting with a nucleicacid sequence contained in a gene encoding an LSR receptor, the saidmethod being characterized in that it comprises bringing a host cellexpressing an LSR receptor or a fragment of the said receptor intocontact with a candidate compound capable of modifying the expression orthe regulation of the expression of the said nucleic sequence, anddetecting, directly or indirectly, a modification of the expression orof the activity of the LSR receptor.

[0068] The invention also relates to a method for selecting chemical orbiochemical compounds capable of interacting with the LSR receptor, thesaid method being characterized in that it comprises bringing an LSRreceptor or a fragment of the said receptor, or a host cell expressingan LSR receptor or a fragment of the said receptor, into contact with acandidate compound capable of modifying the LSR activity, and detecting,directly or indirectly, a modification of the activity of the LSRreceptor or the formation of a complex between the candidate compoundand the said LSR receptor or the said polypeptide.

[0069] The invention comprises the compounds capable of interactingdirectly or indirectly with an LSR receptor as well as the compoundscapable of interacting with one or more nucleic sequences of the LSRreceptor. It also comprises the chemical or biochemical compoundsallowing the expression or the activity of the receptor according to theinvention to be modulated. The compounds, characterized in that theywere selected by one of the methods according to the present invention,also form part of the invention.

[0070] In particular, among these compounds according to the invention,there are preferred the antibodies according to the invention, thepolypeptides according to the invention, the nucleic acids,oligonucleotides and vectors according to the invention, or a leptin orone of its derived compounds, preferably one of its protein variants, orleptins which are chemically modified or are obtained by geneticrecombination, or the protein gC1qR or one of its analogues, or one oftheir fragments.

[0071] The invention comprises, finally, compounds capable of modulatingthe expression or the activity of the receptor according to theinvention, as medicament for the prevention of pathologies and/or ofpathogeneses such as obesity and anorexia, hyperlipidemias,atherosclerosis, diabetes, hypertension, and more generally the variouspathologies associated with abnormalities in the metabolism ofcytokines.

DETAILED DESCRIPTION

[0072] The LSR Receptor

[0073] The invention relates to a purified LSR receptor (“LipolysisStimulated Receptor”), preferably hepatic, consisting of at least one αor α′ subunit and at least one β subunit. The α subunit has a molecularweight of about 66 kDa in rats and in mice and of about 72 kDa inhumans. The α′ subunit has a molecular weight of about 64 kDa in ratsand in mice and of about 70 kDa in humans. The β subunit has a molecularweight of about 58 kDa in rats and in mice and of about 64 kDa inhumans.

[0074] The inventors have formulated the hypothesis according to whichthe most abundant, and probably the most active, form of the LSRreceptor is that in which an a or α′ subunit and three β subunits exist.It appears, however, possible that the α and α′ subunits, on the onehand, and the β subunit, on the other, have distinct biologicalfunctions and that these functions can be performed in a cellindependently of their assembly in the form of a receptor.

[0075] The inventors have also observed that a complex can form betweenthe LSR receptor and the gC1qR receptor having a molecular weight ofabout 33 kDa, or a homologous protein. It appears that the gC1qRreceptor is transiently combined with the LSR receptor and that thepresence of a C1q protein or of homologous proteins makes it possiblenot only to dissociate gC1qR from the LSR receptor but also to activatethe LSR receptor, including in the absence of fatty acids.

[0076] Activity of the LSR Receptor and Applications

[0077] The present invention therefore relates to a receptor, inparticular of hepatic cells, characterized in that it is capable, in thepresence of free fatty acids, of binding lipoproteins, and in theabsence of free fatty acids, of binding a cytokine, preferably the boundleptin, lipoproteins and cytokine being incorporated and then degradedby the cell, it being possible for the said receptor, in addition, tobind the gC1qR protein or one of its analogous proteins.

[0078] Clearance of Lipoproteins

[0079] The LSR receptor represents the principal pathway for theelimination of lipoproteins of intestinal origin and of particles highin triglycerides, in particular VLDLs and chylomicrons. The LSR receptorcan also serve as a pathway for the elimination of LDLs, particles highin cholesterol, which are for the most part removed by the LDL receptorpathway, but of which about 30% are eliminated at the hepatic level bypathways different from the LDL receptor.

[0080] The inventors have in fact demonstrated that the LSR receptor iscapable of binding lipoproteins, in particular the lipoproteins high intriglycerides, and then of internalizing and degrading them. Thislipoprotein clearance activity by the receptor requires the presence offree fatty acids, for example oleate, and is inhibited in the presenceof antibodies directed against LSR or against peptides derived from LSR.

[0081] Clearance of cytokines

[0082] The inventors have also demonstrated that in the absence of freefatty acids, for example oleate, the LSR receptor is capable of bindingcytokines, preferably leptin. The leptin clearance function is, however,only possible if the receptor has not bound fatty acids produced by thehepatic lipase or by the hormone-sensitive lipase of the adipose tissue.Once the cytokines have been bound, the LSR receptor internalizes themand degrades them. This cytokine, preferably leptin, degradationactivity is inhibited by antibodies directed against LSR or againstpeptides derived from LSR.

[0083] The inventors have shown that it is the α subunit of the LSRreceptor which is most particularly involved in the binding ofcytokines, and preferably of leptin.

[0084] Furthermore, the inventors have shown, with the aid of mice,that, in vivo, the LSR receptors carry out the hepatic capturing ofcytokines, preferably of leptin.

[0085] The high levels of leptin in all obese human subjects can beexplained by several molecular mechanisms which are capable of reducingthe hepatic clearance of leptin, including in particular:

[0086] a) alteration of one or more genes for LSR, and/or of theirpromoters;

[0087] b) facilitation, by post-transcriptional modifications, of theallosteric rearrangement allowing the passage from thecytokine-competent conformation to the lipoprotein receptorconformation;

[0088] c) deficiency in the transport of vesicles containing LSR from,or towards, the plasma membrane (this function depends on the integrityof the cytoskeleton);

[0089] d) increase in the degradation of LSR;

[0090] e) increase in the lipid calorie ration which, by diverting thereceptor towards the clearance of lipoproteins, reduces in part itscapacity to degrade leptin.

[0091] Control of LSR Activity by the Cytokines

[0092] Finally, the inventors have demonstrated that cytokines,preferably leptin, modulate the activity of the LSR receptor in thepresence of free fatty acids. More particularly, the cytokines increasethe lipoprotein clearance activity of the LSR receptor and moreprecisely, the binding, internalization and degradation of the VLDLs andLDLs. This increase in the LSR activity could be the result of theincrease in the apparent number of LSR receptor at the surface of thecells following an increase in protein synthesis and following amobilization of endocytosis vesicles. In addition, the inventors haveshown, with the aid of mice, that, in vivo, cytokines, preferablyleptin, are capable of reducing postprandial lipaemic response.

[0093] Leptin, and probably other cytokines, are therefore regulators ofthe activity of LSR. A syndrome of resistance to leptin, or to othercytokines, can lead to a hypertriglyceridemia, which is either permanentor limited to the postprandial phase.

[0094] Treatment of Obesity

[0095] The role played by LSR in the clearance of leptin makes itpossible to formulate a physiopathological model which requires arevision of the strategies used for treating obesity. It is indeedessential to reduce the concentrations of leptin in obese human subjectsin order to restore the physiological fluctuations of this hormone.

[0096] Accordingly, it is possible to envisage using compounds for thetreatment of obesity allowing modulation of the number of LSR receptors,of their recycling rate, or of the change in their conformation, and/orallowing in particular:

[0097] 1. leptinemia, and therefore the sensations of satiety and ofhunger, to be controlled;

[0098] 2. normal leptin concentrations to be restored and normalregulation of dietary habit by the normal perception of the sensationsof hunger and of satiety;

[0099] 3. triglyceridernia to be controlled;

[0100] 4. the plasma concentrations of residues of chylomicrons, highlyatherogenic particles, to be regulated.

[0101] The role played by the LSR receptor in the hepatic clearance oflipoproteins of intestinal region makes it possible to envisage usingcompounds capable of modulating the expression and/or the activity ofLSR in order to modulate the distribution of lipids of dietary originbetween the peripheral tissues, in particualr the adipose tissues, andthe liver. A treatment of obesity will consist in promoting the hepaticdegradation of lipoproteins, and thereby reducing their storage in theadipose tissue, and regulating their plasma concentrations. The lattereffect makes it possible to envisage the use of such compounds to reducethe risks associated with obesity, in particular the atherogenic risks.

[0102] Treatments of Anorexia and of Cachexia

[0103] It is possible to envisage using methods of regulating theactivities of LSR to introduce treatments which make it possibile toovercome the vicious circle which characterizes anorexia nervosa. Byreducing the number of receptors, it should be possible to promoteweight gain in anorexic or undernourished subjects.

[0104] Under these conditions, it is advantageous to selectively inhibitthe clearance of leptin by using synthetic peptides or pharmacologicalmolecules which either reduce the synthesis of LSR or block its capacityto bind leptin and/or lipoproteins, or alternatively increase thecatabolism of the receptor. Treatment of Abnormalities in the Metabolismof Cytokines

[0105] Analysis of the primary structure of the α subunit of LSR, asdescribed below, shows a site homologous to the cytokine binding sitespresent on their receptors, as well as two routing signals which allowendocytosis and rapid degradation of ligands in the lysozomes. Thisobservation is new in the sense that the cytokine receptors do not allowthe internalization and the degradation of ligands. These receptors havebeen characterized on the basis of their intracellular signallingproperties.

[0106] Thus, in addition to it having the property of allowing theproteolytic degradation of lipoproteins and of leptin, it is highlyprobable that the LSR receptor also carries out the degradation of othercytokines. This function can be studied by virtue of the anti-LSRantibodies and of transfected CHO cells expressing the α subunit of LSRas described in Example 4. The involvement of LSR in the clearance ofcytokines is essential because these molecules play an important role inthe regulation of the metabolism of lipids, of the metabolism ofglucose, and in the regulation of food intake and of weight gain.

[0107] The molecular mechanisms by which the cytokines modulate thephysiological functions involved in obesity and its complications arenumerous and complex. It is worth noting, however, the fact thatabnormalities in the metabolism of cytokines are associated withhypertriglyceridemia which frequently accompanies viral, bacterial orprotozoal infections. Moreover, cytokines, and more particularly TumorNecrosis Factor (TNF), induce a transient hypertriglyceridemia similarto that observed in certain forms of obesity-related diabetes.

[0108] The reduction in the number of LSR receptors expressed in theliver of obese mice could explain a deficiency in the elimination ofsome cytokines, this deficiency causing metabolic disruptions such asthose found in obesity. The use of hepatic cells in culture, and of thevarious models of obese animals cited below, will make it possible todetermine, among all the cytokines and more particularly those whichinduce weight loss (IL-6, LIF, OSM, CNTF, IL-11, IL-12α, as well as TNFαand TNFβ), those which modulate the expression and/or the activity ofLSR. The determination of such cytokines can, for example, be carriedout using methods such as those presented in Examples 4 to 6.

[0109] Finally, analysis of the primary structure of the a LSR revealspotential phosphorylation sites. This opens the perspective of aregulation of cellular activity by the LSR receptor. A particularlyimportant example would be the involvement of LSR in the regulation ofthe production of “Acute Phase Proteins ” under the impetus of variousstimuli, including cytokines.

[0110] The involvement of LSR in the clearance and the degradation ofcytokines may, in addition, not be limited to the liver. Indeed, whileit has been demonstrated that the expression of LSR is predominantlyhepatic, it is also certain that the expression of this receptor is notlimited to this organ. Preliminary Northern-blot analysis on varioushuman tissues has been able to reveal, in addition to the hepaticproducts, expression products in the kidney and in the testicle. A morethorough analysis will make it possible to show the different tissuesexpressing LSR in humans. In this perspective, LSR could be involved inthe degradation of cytokines not only at the hepatic level, but also atthe level of the peripheral tissues. A deficiency in this activity couldbe involved in the pathogenesis of autoimmune diseases, of multiplesclerosis and of rheumatoid arthritis. Accumulation of cytokines isfrequently found in the pathogenesis of these diseases.

[0111] Polypeptide Sequences of the LSR Receptor

[0112] The invention relates to polypeptides, characterized in that theyare a constituent of an LSR receptor according to the invention. Theinvention relates more particularly to the polypeptides characterized inthat they constitute the a, α′ or β subunits of the LSR receptor.

[0113] The invention relates more particularly to a purified, isolatedor recombinant polypeptide comprising a sequence of at least 5,preferably of at least 10 to 15 consecutive amino acids of an LSRreceptor, as well as the homologues, equivalents or variants of the saidpolypeptide, or one of their fragments. Preferably, the sequence of atleast 10 to 15 amino acids of the LSR receptor is a biologically activefragment of an LSR receptor.

[0114] Preferably, the invention relates to purified, isolated orrecombinant polypeptides comprising a sequence of at least 10 to 15amino acids of a rat LSR receptor, of a mouse LSR receptor or of a humanLSR receptor.

[0115] In a first preferred embodiment of the invention, the polypeptideis characterized in that it comprises a sequence of at least 10 to 15consecutive amino acids of a sequence chosen from the group comprisingthe sequences of SEQ ID 2, SEQ ID 4 and SEQ ID 6, as well as thevariants, equivalents or homologues of this polypeptide, or one of theirfragments. Preferably, the polypeptide is a homologue or a biologicallyactive fragment of one of the abovementioned sequences.

[0116] In a second preferred embodiment of the invention, thepolypeptide is characterized in that it comprises a sequence of at least10 to 15 consecutive amino acids of a sequence chosen from the groupcomprising the sequences of SEQ ID 16, SEQ ID 17 and SEQ ID 18, as wellas the variants, equivalents or homologues of this polypeptide, or oneof their fragments. Preferably, the polypeptide is a homologue or abiologically active fragment of one of the abovementioned sequences.

[0117] In a third preferred embodiment of the invention, the polypeptideis characterized in that it comprises a sequence of at least 10 to 15consecutive amino acids of a sequence chosen from the group comprisingthe sequences of SEQ ID 8, SEQ ID 10 and SEQ ID 12, as well as thevariants, equivalents or homologues of this polypeptide, or one of theirfragments. Preferably, the polypeptide is a homologue or a biologicallyactive fragment of one of the abovementioned sequences.

[0118] Among the preferred polypeptides of the invention, there will benoted particularly the polypeptides having the human sequence SEQ ID 8,SEQ ID 10 or SEQ ID 12, as well as those having the rat sequence SEQ ID2, SEQ ID 4 or SEQ ID 6, or those having the mouse sequence SEQ ID 16,SEQ ID 17 or SEQ ID 18. The fragments corresponding to the domainsrepresented in FIGS. 1 to 6, whose positions on the sequencescorresponding to SEQ ID 2, 8 or 16, are indicated in Tables 1, 3 and 4.

[0119] Finally, the invention also relates to the polypeptides of SEQ ID29 and SEQ ID 30.

[0120] The present invention also relates to polypeptides comprising thepolypeptides described above, as well as their homologous, equivalent orvariant polypeptides, as well as the fragments, preferably biologicallyactive, of the said polypeptides.

[0121] Among the polypeptides according to the invention, also preferredare the polypeptides comprising or consisting of an amino acid sequencechosen from the amino acid sequences as described above, characterizedin that the said polypeptides are a constituent of the receptoraccording to the invention.

[0122] Analysis of the Polypeptide Sequences of the α, α′ and β Subunitsof the LSR Receptor

[0123] The systematic analysis of the products of the 3 rat cDNAsdescribed in the present application is schematically represented inFIG. 1. The α subunit of the rat LSR receptor, a protein encoded by thelonger cDNA (LSR-Rn-2097), has the following characteristics.

[0124] Potential glycosylation sites are found at positions 12-14 and577-579. A potential site of attachment of glycosaminoglycans is foundat position 14-17.

[0125] Several phosphorylation sites are located at the level of theNH₂-terminal end (positions 193-196, 597-600, 169-171, 172-174, 401403,424426, 464466, 467-469, 185-188, 222-225, 436439, 396-399, 504-507,530-533, 624-627, 608-615), suggesting that the latter is orientedtowards the intracellular region.

[0126] Moreover, the protein has, on the NH₂-terminal side, ahydrophobic amino acid sequence separated into two parts by 2 aminoacids inducing a hairpin structure in which the two arms would consistof hydrophobic amino acids. It is reasonable to assume that this regionrepresents the fatty acid binding site of LSR. The glove-fingerstructure thus produced can accommodate an aliphatic hydrocarbon chain.The two amino acids are, more precisely in the case of rat LSR, twoProlines situated at positions 31 and 33 of the polypeptide sequence ofthe α subunit.

[0127] Still on the NH₂-terminal side is a consensus sequence forbinding to clathrin, a protein which lawns the inner surface of the“coated pits ” (Chen et al., 1990). These specific regions of the plasmamembrane allow rapid endocytosis of membrane proteins. Such a consensussequence is found at the level of the LRP-α₂-macroglobulin receptor, ofCRAM and of the LDL receptor (Herz et al., 1988; Lee et al., 1990;Goldstein et al., 1995). The consequence of a mutation at this level isa substantial delay in the internalization of the LDLs and inducesfamilial hypercholesterolemia (Davis et al., 1986).

[0128] The receptor then possesses a hydrophobic amino acid sequencewhich constitutes a potential transmembrane domain. The length of thissegment allows only one passage across the phospholipid bilayer (Brendelet al., 1992).

[0129] Between this clathrin binding signal and the hydrophobic chaincorresponding to the single transmembrane segment are 2 motifs LI et LL(Letourneur et al., 1992). These two motifs are found in the followingproteins: glut 4 glucose carrier (Verhey et al., 1994); the nonvariantchain and the histocompatibility complex class 11 (Zhong et al., 1997Parra-Lopez et al., 1997). These signals control endocytosis and theintracellular addressing of proteins in the peripheral membrane system.

[0130] On the C-terminal side, there is then a cysteine-rich regionwhich exhibits homology with the cytokine receptors and moreparticularly: the TNF 1 and 2 (Tumor Necrosis Factor 1 and 2) receptors;the low-affinity NGF (Nerve Growth factor) receptor; the Shope fibromavirus TNF soluble receptor; CD40, CD27 and CD30, receptors for thecytokines CD40L, CD27L and CD30L; the T cell protein 4-1BB, receptor forthe putative cytokine 4-1BBL, the FAS antigen (APO 1), receptor for theFASL protein involved in apoptosis, the T cell 0×40 antigen, receptorfor the cytokine 0X40L, and the vaccinia virus A53 protein (Cytokinesand their receptors, 1996; Banner et al., 1993).

[0131] In addition to this cysteine-rich segment, there is a region ofamino acids which are alternately charged + and − (Brendel et al.,1992). This region provides a potential binding site for the apoproteinligands Apo B and Apo E.

[0132] This region contains, in addition, an RSRS motif found in larnin(Simos et al., 1994) and in SF2′ (Krainer et al., 1991).

[0133] The LSR α′ form encoded by the LSR-Rn-2040 cDNA possesses all thedomains described above based on the LSR a sequence encoded by theLSR-Rn-2097 cDNA, with the exception of the LI/LL element, whose Leucinedoublet is removed by alternative splicing. Although possessingsequences which are very similar, the subunits a encoded by LSR-Rn-2097and α′ encoded by LSR-Rn-2040 could therefore differ in their recyclingrate and their addressing. The β form encoded by LSR-Rn-1893 does notpossess a transmembrane domain or a region rich in cysteines andhomologous to the cytokine receptors. However, it possesses at theNH₂-terminal level the hydrophobic region separated by a repetition ofprolines, the region rich in charged amino acids and the RSRS motif.This constituent is probably positioned entirely outside the cell whereit is bound via disulphide bridges either to the product of LSR-Rn-2040,or to that of LSR-Rn-2097.

[0134] Table 1 below lists the different domains or motifs describedabove, indicates whether or not they belong to each of the subunits ofthe LSR receptor, as well as the positions of the start and end of thesaid domains or motifs, or of regions carrying the said domains ormotifs, as indicated in the sequence of SEQ ID 2. TABLE 1 Position onSEQ ID 2 Presence on: Domain or motif Start End α α′ β Potential fattyacid binding site 23 41 X X X Potential clathrin binding site 104 107 XX X Signal for transport: LI 183 184 X X X            LL 195 196 XTransmembrane domain 204 213 X X Potential cytokine receptor site 214249 X X RSRS motif 470 473 X X X Potential lipoprotein ligand bindingsite 544 557 X X X

[0135] Comparison of the Polypeptide Sequences of the LSR Receptors inRats, Mice and Humans

[0136] The lengths of the polypeptide sequences, as well as the SEQ IDsof their respective sequences in the listing included, of the threetypes of subunit of the LSR receptors according to the invention, inrats, mice and humans, are indicated in Table 2a below. TABLE 2aPolypeptide Rat Mouse Human α subunit 593 aa (SEQ ID 2) 594 aa (SEQ ID16) 649 aa (SEQ ID 8) α′ subunit 574 aa (SEQ ID 4) 575 aa (SEQ ID 17)630 aa (SEQ ID 10) β subunit 525 aa (SEQ ID 6) 526 aa (SEQ ID 18) 581 aa(SEQ ID 12)

[0137] These polypeptide sequences were obtained from each of the threecorresponding cDNA sequences, in rats, mice and humans, which will bedescribed in detail later. These polypeptide sequences were obtainedfrom each of the three corresponding cDNA sequences, in rats, mice andhumans, which will be described in detail later. The nomenclature usedto designate these cDNA sequences, which reflects their length in termsof nucleotides, as well as the SEQ IDs of their respective sequences inthe listing included, are presented in Table 2b below. TABLE 2b cDNAcRat Mouse Human α subunit LSR-Rn-2097 LSR-Mm-1886 LSR-Hs-2062 (SEQ ID 1)(SEQ ID 13) (SEQ ID 7) α′ subunit LSR-Rn-2040 LSR-Mm-1829 LSR-Hs-2005(SEQ ID 3) (SEQ ID 14) (SEQ ID 9) β subunit LSR-Rn-1893 LSR-Mm-1682LSR-Hs-1858 (SEQ ID 5) (SEQ ID 15) (SEQ ID 11)

[0138] The protein sequence, corresponding to the α subunit of the LSRreceptor, deduced from the LSR-Hs-2062 sequence has a length of 649amino acids. It is aligned with the protein sequences deduced fromLSR-Mm-1886, 594 amino acids long, and from LSR-Rn-2097, 593 amino acidslong (FIGS. 2A and 2B). The conservation of the protein sequences isvery high (respectively 80.2% and 82.2% identity for 591 and 590overlapping amino acids). The functional domains identified in theprotein sequence of the rat LSR α are found in the human LSR a sequenceas well as in that of the murine LSR a (FIGS. 2A and 2B).

[0139] The human proteins corresponding to the LSR-Hs-2005 (α′) andLSR-Hs-1858 (β) forms have a predicted size of 630 and 581 amino acidsrespectively. The rat proteins corresponding to the LSR-Rn-2040 (α′) andLSR-Rn-1893 (β) forms have a predicted size of 574 and 525 amino acidsrespectively. The mouse proteins corresponding to the LSR-Mm-1829 (α′)and LSR-Mm-1682 (β) forms have a predicted size of 575 and 526 aminoacids respectively. The alignment of the three human forms (FIGS. 3A and3B), of the three forms described in rats (FIGS. 4A and 4B) and of thethree forms described in mice (FIGS. 5A and 5B) shows that in the threespecies, all the protein forms conserve the NPGY signal for binding toclathrin and the RSRS motif. The human (product of LSR-Hs-2062), rat(product of LSR-Rn-2097) and mouse (product of LSR-Mm-1886) long forms(α) exhibit all the functional characteristics of LSR. The three shortforms (β) (respective products of LSR-Hs-1817, LSR-Rn-1893 andLSR-Mm-1682) lose the di-leucine domain for lysosomal addressing, thetransmembrane domain and the cytokine receptor signature. It is alsopossible to observe that the three intermediate forms (α′) (product ofLSR-Hs-2005, of LSR-Rn-2040 and LSR-Mn-1829) lose the di-leucin domain,the transmembrane domain and the domain corresponding to the cytokinereceptor signature being conserved (FIGS. 3A, 3B, 4A, 4B, 5A and 5B).FIG. 6 finally represents the proteins derived from the three cDNA formsidentified in humans, and the motifs carried by each of them as a resultof the splicing from which each is derived.

[0140] Table 3 below lists the different domains or motifs describedabove, as well as the positions of the start and end of the said domainsor motifs, or of regions carrying the said domains or motifs, asindicated in the mouse SEQ ID 16 sequence. TABLE 3 Position on SEQ ID 16Presence on: Domain or motif Start End α α′ β Potential fatty acidbinding site 23 41 X X X Potential clathrin binding site 104 107 X X XSignal for transport: LI 183 184 X X X            LL 195 196 XTransmembrane domain 204 213 X X Potential cytokine receptor site 214249 X X RSRS motif 470 473 X X X Potential lipoprotein ligand bindingsite 544 558 X X X

[0141] Table 4 below lists the different domains or motifs describedabove, as well as the positions of the start and end of the said domainsor motifs, or of regions carrying the said domains or motifs, asindicated in the human SEQ ID 8 sequence. TABLE 4 Position on SEQ ID 8Presence on: Domain or motif Start End α α′ β Potential fatty acidbinding site 76 94 X X X Potential clathrin binding site 157 160 X X XSignal for transport: LI 236 237 X X X            LL 248 249 XTransmembrane domain 257 266 X X Potential cytokine receptor site 267302 X X RSRS motif 527 530 X X X Potential lipoprotein ligand bindingsite 601 613 X X X

[0142] In conclusion, the similarity in the sequence and structure ofLSR which is described above makes it possible to extrapolate to humansthe observations made in rats and/or mice.

[0143] Homologous polypeptide will be understood to mean thepolypeptides exhibiting, compared with the natural polypeptide, certainmodifications such as in particular a deletion, truncation, extension,chimeric fusion and/or mutation, in particular a point mutation. Amongthe homologous polypeptides, those in which the amino acid sequenceexhibits at least 80%, preferably 90%, homology with the amino acidsequences of the polypeptides according to the invention are preferred.

[0144] Equivalent polypeptide will be understood to mean a polypeptidehaving at least one of the activities of the LSR receptor, in particularthe activity of the receptor for lipoproteins or chylomicrons, theactivity of the receptor for cytokine, in particular leptin, or theactivity of the receptor for the gC1q-R protein or one of its analogousproteins. Equivalent polypeptide will also be understood to mean anypolypeptide resulting from the alternative splicing of the genomicnucleic sequence encoding the polypeptides according to the invention.

[0145] Variant polypeptide (or protein variant) will be understood tomean all the mutated polypeptides which may exist, in particular inhuman beings, and which correspond in particular to truncations,deletions and/or additions of amino acid residues, substitutions ormutations, in particular point mutations, as well as the artificialvariant polypeptides which will nevertheless be called variantpolypeptides. In the present case, the variant polypeptides will be inparticular partly associated with the onset and with the development ofobesity or anorexia. They may also be associated with the onset and/ordevelopment of the risks or complications associated with obesity, inparticular at the cardiovascular level, and/or of pathologies associatedwith abnormalities in the metabolism of cytokines.

[0146] Polypeptide fragment is understood to mean a polypeptide or apeptide encoded by a nucleic sequence comprising a minimum of 15nucleotides or bases, preferably 20 bases or 30 bases. These fragmentsmay comprise in particular a point mutation, compared with the normalpolypeptide sequence, or may correspond to specific amino acid sequencesof variant polypeptides, artificial or existing in humans, such as thoselinked to a polymorphism linked in particular to obesity or to theabovementioned pathologies.

[0147] Biologically active fragment will be understood to mean inparticular a fragment of an amino acid sequence of a polypeptide:

[0148] exhibiting at least one of the LSR receptor activities, inparticular the lipoprotein receptor activity, or the cytokine,particularly leptin, receptor activity and/or cell signalling activity,and/or

[0149] capable of being recognized by an antibody specific for thereceptor according to the invention, and/or

[0150] capable of being recognized by a compound capable, for example byneutralizing the binding of a ligand specific for the said receptor, ofmodulating the activity of the LSR receptor, and/or

[0151] capable of modulating the addressing and/or cellular location ofthe LSR receptor, and/or

[0152] more generally constituting a biologically active domain or motifof the LSR receptor.

[0153] Among the preferred biologically active fragments according tothe invention, there are in particular:

[0154] the fragments comprising a clathrin binding site,

[0155] the fragments comprising a fatty acid binding site, in particulara fatty acid binding site comprising a hydrophobic amino acid sequenceseparated into two parts by two contiguous prolines, which induce ahairpin structure whose arms consist of hydrophobic amino acids,

[0156] the fragments comprising a hydrophobic region constituting atransmembrane domain,

[0157] the fragments comprising a region capable of controllingendocytosis and intracellular addressing of the proteins in theperipheral membrane system, in particular a fragment comprising a sitecontaining the LI and LL motifs,

[0158] the fragments comprising a cytokine binding site, in particular asite including a cysteine-rich region,

[0159] the fragments comprising a region defining a potential bindingsite for lipoprotein ligands such as ApoB and ApoE, in particular aregion comprising a sequence of amino acids alternately charged + and −,and

[0160] the fragments comprising an RSRS motif.

[0161] There are in particular among these fragments polypeptides asdefined in Tables 1, 2 and 4, or any fragments of the nucleotides of SEQID 2, 8 or 16, comprising the said polypeptides, and any equivalent,homologous or variant fragments.

[0162] Other preferred fragments include antigenic peptides such asthose having the sequences SEQ ID 29 and 30.

[0163] Nucleotide Sequences of the LSR Receptor

[0164] The subject of the present invention is isolated nucleic acidsequences, characterized in that they encode an LSR receptor or apolypeptide according to the invention.

[0165] More particularly, the invention relates to a purified nucleicacid, characterized in that it comprises at least 8, preferably at least10 and more particularly at least 15 consecutive nucleotides of thepolynucleotide of SEQ ID 19, as well as the nucleic acid sequencescomplementary to this nucleic acid.

[0166] The invention also relates to a purified nucleic acid,characterized in that it comprises at least 8, preferably at least 10and more particularly at least 15 consecutive nucleotides of thepolynucleotide of SEQ ID 41, as well as the nucleic acid sequencescomplementary to this nucleic acid.

[0167] The invention also relates to a purified nucleic acid encodingthe human LSR receptor, characterized in that it comprises a nucleotidesequence corresponding to nucleotides 1898 to 21094, particularly tonucleotides 2001 to 20979, more particularly to nucleotides 2145 to20979 of SEQ ID 19, as well as the nucleic acid sequences complementaryto this nucleic acid.

[0168] The invention also relates to the nucleic acid sequencescontained in the gene encoding the human LSR receptor, in particulareach of the exons of the said gene or a combination of exons of the saidgene, or alternatively a polynucleotide extending over a portion of oneor more exons. Preferably, these nucleic acids encode one or morebiologically active fragments of the human LSR receptor.

[0169] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence correspondingto nucleotides 1 to 1897 of SEQ ID 19, as well as the nucleic acidsequences complementary to this nucleic acid.

[0170] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence correspondingto nucleotides 21095 to 22976 of SEQ ID 19, as well as the nucleic acidsequences complementary to this nucleic acid.

[0171] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence chosen from thegroup comprising the sequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, aswell as the nucleic acid sequences complementary to this nucleic acid.

[0172] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence chosen from thegroup comprising the sequences of SEQ ID 1, SEQ ID 3 and SEQ ID 5, aswell as the nucleic acid sequences complementary to this nucleic acid.

[0173] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence chosen from thegroup comprising the sequences of SEQ ID 13, SEQ ID 14 and SEQ ID 15, aswell as the nucleic acid sequences complementary to this nucleic acid.

[0174] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence correspondingto nucleotides 1898 to 2001 of SEQ ID 19 or preferably to nucleotides1898 to 2144 of SEQ ID 19, as well as the nucleic acid sequencescomplementary to this nucleic acid.

[0175] The invention also relates to a purified nucleic acid,characterized in that it comprises a nucleotide sequence correspondingto nucleotides 20980 to 21094 of SEQ ID 19, as well as the nucleic acidsequences complementary to this nucleic acid.

[0176] Among the nucleic acids according to the invention, the nucleicacids having the nucleotide sequence chosen from the group comprisingthe sequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, the sequences of SEQID 1, SEQ ID 3 and SEQ ID 5, as well as the sequences of SEQ ID 13, SEQID 14 and SEQ ID 15, as well as their complementary sequences, arepreferred.

[0177] Also forming part of the invention are the variant, mutated,equivalent or homologous sequences of the sequences according to theinvention, as well as their fragments and the nucleic sequences capableof hybridizing specifically with the sequences according to theinvention.

[0178] Human Genomic Sequence

[0179] The invention therefore relates to the genomic sequence of thehuman LSR receptor, preferably the sequence of SEQ ID 19, as well astheir complementary sequences or one of their allelic variants, themutated, equivalent or homologous sequences, or one of their fragments.

[0180] The gene for human LSR (SEQ ID 19) comprises 10 exons distributedover 21 094 bp. The size of the exons is respectively: 356, 345, 120,57, 147, 174, 60, 132, 626 and 141 bp (Table 5). TABLE 5 EXON START END5′ SPLIC. BL 5′ 3′ SPLIC. BL 3′ Ex1 1898 2253 — — GTACGG +2 Ex2 34373781 CAG +1 GTATGT +1 Ex3 12067 12186 CAG +2 GTGAGT +1 Ex4 15047 15103CAG +2 GTACGG +1 Ex5 15668 15814 CAG +2 GTAAGT +1 Ex6 19481 19654 CAG +2GTGAGG +1 Ex7 19801 19860 CAG +2 GTGAGA +1 Ex8 19958 20089 TAG +2 GTAAGC+1 Ex9 20231 20856 CAG +2 GTGAGG   0 Ex10 20946 21094 CAG   0 —

[0181] EXON column indicates the exons numbered from 1 to 10 in the5′-3′ order of their position on the genomic sequence. The START and ENDcolumns indicate respectively the position of the first and of the lastnucleotide of the exon considered. The sequences of the splicing sitebordering the exon in 5′ and 3′ are indicated in the 5′SPLIC and 3′SPLICcolumns. The BL 5′ and BL 3′ columns indicate the number of bases in 5′and in 3′, respectively, of an exon which will be used in the readingframe of the messenger only after splicing. For example: as exon 7 has afree base in 3′, this exon can be joined to the 5′ end of exon 8 whichhas 2 free bases in 5′. The combination 1 base+2 bases constitutes thecodon which was destroyed by the intron in the genomic sequence. Exon 7may be joined by its 3′ end to any exon having two free bases in 5′; ifthe new codon created does not correspond to a stop codon, the openreading frame will be conserved.

[0182] Exons 1 and 2 as well as 9 and 10 are necessarily co-spliced,thus forming a 5′ block corresponding to exons 1 and 2 and a 3′ blockcorresponding to exons 9 and 10. The functional minimal messenger,corresponding to the product of these four exons, could therefore have asize of about 1 331 bp. For the other exons, all the possiblecombinations make it possible to conserve the open reading frame.

[0183] The size of the noncoding exons in 5′ could not be determinedwith precision. Indeed, the rat 5′ UTR sequences are too divergent fromthose of humans to finalize the analysis of these sequences and toidentify the real 5′ end of the human LSR cDNA. This can be carried outby isolating the 5′ end of the human LSR messengers by the 5′ endcapture methods developed by the inventors (WO 96/34981). Thepolyadenylation site described below is the only one which is presentbefore the USF2 gene, situated in 3′ of the human LSR gene. It istherefore very likely that the untranslated 3′ region of this gene isvery short (of an estimated size of about 100 bp). All the sizes givenin relation to the human LSR cDNA molecules will therefore have to beadjusted according to the size of the untranslated 5′ end. The humancDNA sequence obtained taking into account all the exons deduced fromthe analysis of the genomic sequence have a size of 2 158 bp. This formcould correspond to the LSR-Rn-2097 form.

[0184] The location of some of the signals for expression of thenucleotide sequence of SEQ ID 19 is presented in Table 6 which follows.TABLE 6 Signal Start End preferred ATG 2145 2147 other possible ATG 20012003 STOP 20977 20979 POLY Ad 21065 21070

[0185] The characteristic elements of the messenger RNA molecule aredescribed in the Signal column: Initiation of translation (ATG),termination of translation (STOP) and polyadenylation signal (POLY Ad).The Start and End columns indicate the position as nucleotide for thestart and end of these signals on the genomic sequence SEQ ID 19. An ATGsignal for initiation of translation is preferred to another because itprovides an environment which is more suitable for initiation.

[0186] The present invention also relates to the purified nucleic acidsequences encoding one or more elements for regulating the expression ofthe human LSR gene. Also included in the invention are the nucleic acidsequences of the promoter and/or regulator of the gene encoding thereceptor according to the invention, or one of their allelic variants,the mutated, equivalent or homologous sequences, or one of theirfragments.

[0187] The invention relates more particularly to a purified nucleicacid situated in 5′ of the coding sequence of the LSR gene. This nucleicacid is characterized in that it comprises a nucleotide sequencecorresponding to nucleotides 1 to 1897 of SEQ ID 19, as well as thenucleic acid sequences complementary to this nucleic acid. Shorterfragments of this nucleic acid may also be used as promoters forexpression of the LSR gene or of any other sequence encoding aheterologous polypeptide.

[0188] The invention also relates to a purified nucleic acid situated in3′ of the transcribed sequence of the LSR gene. This nucleic acid ischaracterized in that it comprises a nucleotide sequence correspondingto nucleotides 21095 to 22976 of SEQ ID 19, as well as the nucleic acidsequences complementary to this nucleic acid. Shorter fragments of thisnucleic acid can also be used as elements regulating the expression ofgenes.

[0189] Finally, the invention also relates to the genomic sequence ofthe human LSR receptor, preferably the sequence of SEQ ID 41, as well astheir complementary sequences, or one or their allelic variants, themutated, equivalent or homologous sequences, or one of their fragments.

[0190] Comparison of the Genomic Organizations in Humans, Rats and Mice

[0191] It is advantageous to note that a syntheny (conservation of theorganization of certain chromosomal regions between species) between themouse chromosome 7 region where the Lisch7 gene is located, in theimmediate vicinity of USF2, and the human chromosome 19 region 19q13,carrying LSR, is well described. The organization of the two Lisch7/LSRand USF2 genes is conserved between species. Likewise, Apo E, which isof a more centromeric location relative to these genes, exists both inmice and in humans. It is remarkable that the LSR lipoprotein receptorand one of their ligands ApoE are located in the same chromosomalregion. Indeed, the receptor and the ligand are frequently co-regulated.Such a situation would make it possible to envisage that the phenomenaobserved in mice are applicable to humans.

[0192] Human, Rat and Mouse cDNA Sequences

[0193] The invention relates, in addition, to 3 different cDNAs derivedfrom the LSR receptor gene by alternative splicing. These 3 cDNAs havebeen identified in humans, rats and mice (Table 2b). They encode thethree types of LSR receptor subunits, α (long), α′ (intermediate) and β(short). The longest cDNA contains the totality of the 10 exons of thegene. The intermediate cDNA does not comprise exon 4. Finally, theshortest cDNA does not contain exons 4 and 5.

[0194] The human LSR-Hs-2062 cDNA nucleotide sequence, encoding the αsubunit of the LSR receptor, and the rat LSR-Rn-2097 cDNA nucleotidesequence are 78.6% identical over 1 955 bp which overlap. These figuresare respectively 78.8% and 1 851 bp when the murine LSR-Mm-1886 sequence(long form) is aligned with the human sequence. This reflects a veryhigh conservation of the nucleic sequences between species. The highestdivergence levels are observed in the untranslated 5′ end (when thesequence is available), in the first coding exon and in the untranslated3′ end (FIGS. 7 A, 7B, 7C, 7D and 7E).

[0195] The invention therefore also relates to a purified nucleic acid,characterized in that it is chosen from the group comprising thesequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, the sequences SEQ ID 1,SEQ ID 3 and SEQ ID 5, and the sequences SEQ ID 13, SEQ ID 14 and SEQ ID15, as well as the nucleic acid sequences complementary to this nucleicacid, or one of their allelic variants, the mutated, equivalent orhomologous sequences, or one of their fragments.

[0196] The nucleic acids constituting the coding frames of theabovementioned nucleic acids, between the codons for initiation and fortermination of translation, also form part of the invention.

[0197] The nucleic acids encoding the polypeptide fragments according tothe invention are also part of the invention. It will be particularlynoted that the nucleic acids encode the fragments described in Tables 1,3 and 4.

[0198] Thus, Table 7 describes the position of such nucleic acidfragments on the human sequence of SEQ ID 7. TABLE 7 Position on thecDNA of SEQ 7 Domain or motif Start End Potential fatty acid bindingsite 329 385 Potential clathrin binding site 572 583 Signal fortransport: LI 809 814            LL 845 850 Transmembrane domain 872 901Potential cytokine receptor site 902 1009 RSRS motif 1682 1693 Potentiallipoprotein ligand binding site 1904 1942

[0199] The invention also relates to a purified nucleic acidcorresponding to the sequence of the 5′UTR of the cDNAs encoding thehuman LSR receptor. This nucleic acid is characterized in that itcomprises a nucleotide sequence corresponding to nucleotides 1898 to2001 of SEQ ID 19 or preferably to nucleotides 1898 to 2144 of SEQ ID19, as well as the nucleic acid sequences complementary to this nucleicacid. Shorter fragments of this nucleic acid can also be used.

[0200] The invention also relates to a purified nucleic acidcorresponding to the sequence of the 3′UTR of the cDNAs encoding the LSRreceptor. This nucleic acid is characterized in that it comprises anucleotide sequence corresponding to nucleotides 20980 to 21094 of SEQID 19, as well as the nucleic acid sequences complementary to thisnucleic acid. Shorter fragments of this nucleic acid can also be used.

[0201] The invention also relates to the purified nucleic acidscorresponding respectively to the sequences of the 5′UTR or of the 3′UTRof the cDNAs encoding the rat or mouse LSR receptor. Shorter fragmentsof this nucleic acid can also be used.

[0202] The 5′UTR and 3′UTR may contain elements (“responsive elements”and “enhancers ”) which are involved in the regulation of transcriptionand of translation. These regions have in particular a role in thestability of the mRNAs. Furthermore, the 5′UTR comprises theShine-Delgarno motif which is essential for the translation of the mRNA.

[0203] Nucleic acid, nucleic sequence or nucleic acid sequence areunderstood to mean an isolated natural, or a synthetic, DNA and/or RNAfragment comprising, or otherwise, non-natural nucleotides, designatinga precise succession of nucleotides, modified or otherwise, allowing afragment, a segment or a region of a nucleic acid to be defined.

[0204] Equivalent nucleic sequences are understood to mean nucleicsequences encoding the polypeptides according to the invention takinginto account the degeneracy of the genetic code, the complementary DNAsequences and the corresponding RNA sequences, as well as the nucleicsequences encoding the equivalent polypeptides.

[0205] Homologous nucleic sequences are understood to mean the nucleicsequences encoding the homologous polypeptides and/or the nucleicsequences exhibiting a level of homology of at least 80%, preferably90%. According to the invention, the homology is only of the statisticaltype, which means that the sequences have a minimum of 80%, preferably90%, of nucleotides in common. They are preferably sequences capable ofhybridizing specifically with a sequence of the invention. Preferably,the specific hybridization conditions will be like those found in theexamples, or such that they ensure at least 95% homology.

[0206] The length of these nucleic sequences for hybridization can varyfrom 8, 10, 15, 20 or 30 to 200 nucleotides, particularly from 20 to 50nucleotides, more particularly from 20 to 30 nucleotides.

[0207] Allele or allelic variant will be understood to mean the naturalmutated sequences corresponding to polymorphisms present in human beingsand, in particular, to polymorphisms which can lead to the onset and/orto the development of obesity or of anorexia. These polymorphisms canalso lead to the onset and/or to the development of risks orcomplications associated with obesity, in particular at thecardiovascular level, and/or of pathologies associated withabnormalities in the metabolism of cytokines.

[0208] Mutated nucleic sequences are understood to mean the nucleicsequences comprising at least one point mutation compared with thenormal sequence.

[0209] While the sequences according to the invention are in generalnormal sequences, they are also mutated sequences since they comprise atleast one point mutation and preferably at most 10% of mutationscompared with the normal sequence.

[0210] Preferably, the present invention relates to mutated nucleicsequences in which the point mutations are not silent, that is to saythat they lead to a modification of the amino acid encoded in relationto the normal sequence. Still more preferably, these mutations affectamino acids which structure the LSR complex and/or receptor or thecorresponding domains and fragments thereof. These mutations may alsoaffect amino acids carried by the regions corresponding to the receptorsites, for lipoproteins or cytokines, in particular leptin, or to sitesfor binding of cofactors, in particular or free fatty acids, oralternatively to phosphorylation sites. These mutations may also affectthe sequences involved in the transport, addressing and membraneanchorage of LSR.

[0211] In general, the present invention relates to the normal LSRpolypeptides, the mutated LSR polypeptides as well as fragments thereofand to the corresponding DNA and RNA sequences, the LSR polypeptidesdesignating polypeptides of the receptor according to the invention.

[0212] According to the invention, the fragments of nucleic sequencesmay in particular encode domains of receptors and polypeptidespossessing a function or a biological activity as defined above, containdomains or regions situated upstream or downstream of the codingsequence and containing elements for regulating the expression of theLSR gene or alternatively possessing a sequence allowing their use as aprobe or as a primer in methods of detection, identification oramplification of nucleic sequences. These fragments preferably have aminimum size of 8, of 10 bases, and fragments of 20 bases, andpreferably of 30 bases, will be preferred.

[0213] Among the nucleic fragments which may be of interest, inparticular for diagnosis, there should be mentioned, for example, thegenomic intron sequences of the gene for the LSR complex, such as inparticular the joining sequences between the introns and the exons,normal or mutated.

[0214] The nucleic acid sequences which can be used as sense orantisense oligonucleotides, characterized in that their sequences arechosen from the sequences according to the invention, also form part ofthe invention.

[0215] Among the nucleic acid fragments of interest, there should thusbe mentioned, in particular the antisense oligonucleotides, that is tosay whose structure ensures, by hybridization with the target sequence,inhibition of the expression of the corresponding product. There shouldalso be mentioned the sense oligonucleotides which, by interaction withthe proteins involved in the regulation of the expression of thecorresponding product, will induce either inhibition, or activation ofthis expression.

[0216] The sequences carrying mutations which may be involved in thepromoter and/or regulatory sequences of the genes for the LSR complex,which may have effects on the expression of the corresponding proteins,in particular on their level of expression, also form part of thepreceding sequences according to the invention.

[0217] The nucleic sequences which can be used as primer or probe,characterized in that their nucleic sequence is a sequence of theinvention, also form part of the invention.

[0218] The present invention relates to all the primers which may bededuced from the nucleotide sequences of the invention and which maymake it possible to detect the said nucleotide sequences of theinvention, in particular the mutated sequences, using in particular amethod of amplification such as the PCR method, or a related method.

[0219] The present invention relates to all the probes which may bededuced from the nucleotide sequences of the invention, in particularsequences capable of hybridizing with them, and which may make itpossible to detect the said nucleotide sequences of the invention, inparticular to discriminate between the normal sequences and the mutatedsequences.

[0220] The invention also relates to the use of a nucleic acid sequenceaccording to the invention as a probe or a primer for the detectionand/or the amplification of a nucleic acid sequence according to theinvention.

[0221] All the probes and primers according to the invention may belabelled by methods well known to persons skilled in the art, in orderto obtain a detectable and/or quantifiable signal.

[0222] The present invention also relates to the nucleotide sequenceswhich may comprise non-natural nucleotides, in particularsulphur-containing nucleotides, for example, or nucleotides of α or βstructure.

[0223] The present invention relates, of course, to both the DNA and RNAsequences, as well as the sequences which hybridize with them, as wellas the corresponding double-stranded DNAs.

[0224] In the text which follows, the preceding DNA sequences will becalled genes for the LSR complex, whether they are normal or pathologicsequences.

[0225] It should be understood that the present invention does notrelate to the genomic nucleotide sequences in their natural chromosomalenvironment, that is to say in the natural state. They are sequenceswhich have been isolated, that is to say that they have been collecteddirectly or indirectly, for example by copying (cDNA), their environmenthaving been at least partially modified.

[0226] Thus, this may also be both cDNA and genomic DNA, partiallymodified or carried by sequences which are at least partially differentfrom the sequences carrying them naturally.

[0227] These sequences may also be termed non-natural.

[0228] The invention also comprises methods for screening cDNA andgenomic DNA libraries, for the cloning of the isolated cDNAs, and/or thegenes coding for the receptor according to the invention, and for theirpromoters and/or regulators, characterized in that they use a nucleicsequence according to the invention. Among these methods, there may bementioned in particular:

[0229] the screening of cDNA libraries and the cloning of the isolatedcDNAs (Sambrook et al., 1989; Suggs et al., 1981; Woo et al., 1979),with the aid of the nucleic sequences according to the invention,

[0230] the screening of 5′ end tag libraries (WO 96/34981) for nucleicsequences according to the invention, and thus the isolation of tagsallowing the cloning of complete cDNAs and the corresponding promotersfrom genomic DNA libraries,

[0231] the screening of genomic libraries, for example of BACs,(Chumakov et al., 1992; Chumakov et al., 1995) and, optionally, agenetic analysis by FISH (Cherif et al., 1990) with the aid of sequencesaccording to the invention, allowing isolation and chromosomal location,and then the complete sequencing of the genes encoding the LSR receptor.

[0232] Also included in the invention is a sequence, in particular agenomic sequence encoding a receptor or a polypeptide according to theinvention, or a nucleic acid sequence of a promoter and/or regulator ofa gene encoding a receptor or a polypeptide according to the invention,or one of their allelic variants, a mutated, equivalent or homologoussequence, or one of their fragments, characterized in that it is capableof being obtained by one of the preceding methods according to theinvention, or a sequence capable of hybridizing with the said sequences.

[0233] Vectors, Host Cells and Transgenic Animals

[0234] The invention also comprises the cloning and/or expressionvectors containing a nucleic acid sequence according to the invention.

[0235] The vectors according to the invention, characterized in thatthey comprise the elements allowing the expression and/or the secretionof the said sequences in a host cell, also form part of the invention.

[0236] The vectors characterized in that they comprise a promoter and/orregulator sequence according to the invention, or a sequence forcellular addressing according to the invention, or one of theirfragments, also form part of the invention.

[0237] The said vectors will preferably comprise a promoter, signals forinitiation and termination of translation, as well as appropriateregions for regulation of transcription. They must be able to be stablymaintained in the cell and may optionally possess particular signalsspecifying the secretion of the translated protein.

[0238] These different control signals are chosen according to thecellular host used. To this end, the nucleic acid sequences according tothe invention may be inserted into autonomously replicating vectorsinside the chosen host, or integrative vectors of the chosen host.

[0239] Among the autonomously replicating systems, there will bepreferably used according to the host cell, systems of the plasmid orviral type, it being possible for the viral vectors to be in particularadenoviruses (Perricaudet et al., 1992), retroviruses, poxviruses orherpesviruses (Epstein et al., 1992). Persons skilled in the art knowthe technologies which can be used for each of these systems.

[0240] When the integration of the sequence into the chromosomes of thehost cell is desired, it will be possible to use, for example, systemsof the plasmid or viral type; such viruses will be, for example,retroviruses (Temin, 1986), or AAVs (Carter, 1993).

[0241] Such vectors will be prepared according to the methods commonlyused by persons skilled in the art, and the clones resulting therefrommay be introduced into an appropriate host by standard methods such as,for example, lipofection, electroporation or heat shock.

[0242] The invention comprises, in addition, the host cells, inparticular eukaryotic and prokaryotic cells, transformed by the vectorsaccording to the invention, as well as transgenic animals, excepthumans, comprising one of the said transformed cells according to theinvention.

[0243] Among the cells which can be used for these purposes, there mayof course be mentioned bacterial cells (Olins and Lee, 1993), but alsoyeast cells (Buckholz, 1993), as well as animal cells, in particularmammalian cell cultures (Edwards and Aruffo, 1993), and in particularChinese hamster ovary cells (CHO), but also insect cells in which it ispossible to use methods using baculoviruses, for example (Luckow, 1993).A preferred cellular host for the expression of the proteins of theinvention consists of the CHO cells.

[0244] Among the mammals according to the invention, there will bepreferred animals such as mice, rats or rabbits, expressing apolypeptide according to the invention, the phenotype corresponding tothe normal or variant LSR receptor, in particular mutated of humanorigin.

[0245] Among the animal models more particularly of interest here, thereare in particular

[0246] transgenic animals exhibiting a deficiency in one of thecomponents of LSR. They are obtained by homologous recombination onembryonic stem cells, transfer of these stem cells to embryos, selectionof the chimeras affected at the level of the reproductive lines, andgrowth of the said chimeras;

[0247] transgenic mice overexpressing one or more of the genes for theLSR complex of murine and/or human origin. The mice are obtained bytransfection of multiple copies of the genes for the LSR complex underthe control of a strong promoter of an ubiquitous nature, or selectivefor a type of tissue, preferably the liver;

[0248] transgenic animals (preferably mice) made deficient in one ormore of the genes for the LSR complex, by inactivation with the aid ofthe LOXP/CRE recombinase system (Rohlmann et al., 1996) or any othersystem for inactivating the expression of a gene at a precise age of theanimal;

[0249] animals (preferably rats, rabbits, mice) overexpressing one ormore of the genes for the LSR complex, after viral transcription or genetherapy;

[0250] crossings of animals deficient in LSR (in particular mice) withanimals deficient in, or overexpressing:

[0251] >the LDL receptor (Herz et al., 1995; Ishibashi et al., 1993)>

[0252] >hepatic lipase (Homanics et al., 1995; Kobayashi et al., 1996)>

[0253] >apoprotein B (Purcellhuynh et al., 1995; Fan et al., 1995)>

[0254] >apoprotein E (Plump et al., 1992; Zhang et al., 1992; Huang etal., 1996)>

[0255] >apoCIII (Aalto-Setälä et al., 1992; Ito et al., 1990; Maeda etal., 1994).

[0256] The production of transgenic animals, and the viral or nonviraltransfections will be preferably carried out on the following rat andmouse lines:

[0257] Zucker rat (fa/fa) (lida et al., 1996)

[0258] AKR/J mouse (West et al., 1992)

[0259] ob/ob mouse (Zhang et al., 1994)

[0260] ob²j/ob²j mouse (ibid)

[0261] tubby mouse (Kleyn et al., 1996; Nobben-Trauth et al., 1996)

[0262] fat/fat (Heldin et al., 1995)

[0263] agouti mouse (Lu et al., 1994; Manne et al., 1995)

[0264] db/db mouse (Chen et al., 1996).

[0265] The cells and mammals according to the invention can be used in amethod for the production of a polypeptide according to the invention,as described below, and can also serve as a model for analysis andscreening.

[0266] The transformed cells or mammals as described above can also beused as models so as to study the interactions between the polypeptidesof the LSR complex, between these and their partners, chemical orprotein compounds, which are involved directly or indirectly in theactivities of the receptor for lipoproteins or the receptor forcytokines, and in particular for leptin, and in order to study thedifferent mechanisms and interactions called into play according to thetype of activity, or according to whether a normal complex is involved,or a complex in which at least one of the domains is a variant.

[0267] In particular, they may be used for the selection of productswhich interact with the LSR complex, or one of its normal or variantdomains, as cofactor or as inhibitor, in particular a competitiveinhibitor, or alternatively having an agonist or antagonist activity onthe conformational changes in the LSR complex. Preferably, the saidtransformed cells will be used as a model allowing, in particular, theselection of products which make it possible to combat obesity or thepathologies mentioned above. The said cells may also serve for thedetection of the potential risks posed by certain compounds.

[0268] Production of Polypeptides Derived From the LSR Receptor

[0269] The invention also relates to the synthesis of synthetic orrecombinant polypeptides of the invention, in particular by chemicalsynthesis or by the use of a nucleic acid sequence according to theinvention.

[0270] The polypeptides according to the present invention can beobtained by chemical synthesis using any of the numerous known peptidesyntheses, for example the techniques using solid phases or techniquesusing partial solid phases, by condensation of fragments or by aconventional synthesis in solution.

[0271] When the compounds according to the present invention aresynthesized by the solid phase method, the C-terminal amino acid isbound to an inert solid support and comprises groups protecting itsamino group at the alpha position (and if necessary, protection on itsfunctional side groups).

[0272] At the end of this step, the group protecting the amino-terminalgroup is removed and the second amino acid, it too comprising thenecessary protection, is bound.

[0273] The N-terminal protecting groups are removed after each aminoacid has been bound; on the other hand, the protection is of coursemaintained on the side chains. When the polypeptide chain is complete,the peptide is cleaved from its support and the side protecting groupsare removed.

[0274] The solid phase synthesis technique is well known to a personskilled in the art. See in particular Stewart et al. (1984) and Bodansky(1984).

[0275] The polypeptides obtained by chemical synthesis and which maycomprise corresponding non-natural amino acids are also included in theinvention.

[0276] The method for the production of a polypeptide of the inventionin recombinant form is itself included in the present invention, and ischaracterized in that the transformed cells, in particular the cells ormammals of the present invention, are cultured under conditions allowingthe expression of a recombinant polypeptide encoded by a nucleic acidsequence according to the invention, and in that the said recombinantpolypeptide is recovered.

[0277] Also forming part of the invention is a method for the productionof a heterologous polypeptide, characterized in that it uses a vector ora host cell containing at least one of the promoter and/or regulatorysequences according to the invention, or at least one of the sequencesfor cellular addressing according to the invention, or one of theirfragments.

[0278] The recombinant polypeptides, characterized in that they arecapable of being obtained by the said method of production, also formpart of the invention.

[0279] The recombinant polypeptides obtained as indicated above may beboth in glycosylated and nonglycosylated form and may or may not havethe natural tertiary structure.

[0280] These polypeptides may be produced from the nucleic acidsequences defined above, according to techniques for the production ofrecombinant polypeptides known to persons skilled in the art. In thiscase, the nucleic acid sequence used is placed under the control ofsignals allowing its expression in a cellular host.

[0281] An effective system of production of a recombinant polypeptiderequires having a vector and a host cell according to the invention.

[0282] These cells may be obtained by introducing into the host cells anucleotide sequence inserted into a vector as defined above, and thenculturing the said cells under conditions allowing the replicationand/or expression of the transfected nucleotide sequence.

[0283] The methods for the purification of a recombinant polypeptidewhich are used are known to persons skilled in the art. The recombinantpolypeptide may be purified from cell lysates and extracts, from theculture medium supernatant, by methods used individually or incombination, such as fractionation, chromatographic methods,immunoaffinity techniques with the aid of specific mono- or polyclonalantibodies, and the like.

[0284] A preferred variant consists in producing a recombinantpolypeptide fused with a “carrier” protein (chimeric protein). Theadvantage of this system is that it allows a stabilization and areduction in proteolysis of the recombinant product, an increase insolubility during in vitro renaturation and/or simplification of thepurification when the fusion partner has affinity for a specific ligand.

[0285] Antibodies

[0286] The mono- or polyclonal antibodies or fragments thereof, chimericor immunoconjugated antibodies, characterized in that they are capableof specifically recognizing a polypeptide or receptor according to theinvention, also form part of the invention.

[0287] Specific polyclonal antibodies may be obtained from a serum of ananimal immunized against, for example:

[0288] the LSR receptor purified from membranes of cells carrying thesaid LSR receptor, by methods well known to persons skilled in the artsuch as affinity chromatography using, for example, recombinant leptinas specific ligand, or

[0289] a polypeptide according to the invention, in particular producedby genetic recombination or by peptide synthesis, according to thecustomary procedures, from a nucleic acid sequence according to theinvention.

[0290] There may be noted in particular the advantage of antibodiesspecifically recognizing certain polypeptides, variants or fragments,which are in particular biologically active, according to the invention.

[0291] The specific monoclonal antibodies may be obtained according tothe conventional hybridoma culture method described by Kohler andMilstein, 1975.

[0292] The antibodies according to the invention are, for example,chimeric antibodies, humanized antibodies, Fab or F(ab′)₂ fragments.They may also be in the form of immunoconjugates or of labelledantibodies so as to obtain a detectable and/or quantifiable signal.

[0293] The invention also relates to methods for the detection and/orpurification of a polypeptide according to the invention, characterizedin that they use an antibody according to the invention.

[0294] The invention comprises, in addition, purified polypeptides,characterized in that they are obtained by a method according to theinvention.

[0295] Moreover, in addition to their use for the purification ofpolypeptides, the antibodies of the invention, in particular themonoclonal antibodies, may also be used for the detection of thesepolypeptides in a biological sample.

[0296] They thus constitute a means for the immunocytochemical orimmunohistochemical analysis of the expression of the polypeptide of theLSR receptor on specific tissue sections, for example byimmunofluorescence, gold labelling, enzymatic immunoconjugates.

[0297] They make it possible in particular to detect abnormal expressionof these polypeptides in the biological tissues or samples, which makesthem useful for the detection of abnormal expression of the LSR receptoror for monitoring the progress of the method of prevention or treatment.

[0298] More generally, the antibodies of the invention may beadvantageously used in any situation where the expression of apolypeptide of the LSR receptor, normal or mutated, needs to beobserved.

[0299] Detection of Allelic Variability and Diagnosis

[0300] Also forming part of the invention are the methods for thedetermination of an allelic variability, a mutation, a deletion, a lossof heterozygosity or a genetic abnormality, characterized in that theyuse a nucleic acid sequence or an antibody according to the invention.

[0301] These methods relate to, for example, the methods for thediagnosis of predisposition to obesity, to the associated risks, or topathologies associated with abnormalities in the metabolism ofcytokines, by determining, in a biological sample from the patient, thepresence of mutations in at least one of the sequences described above.The nucleic acid sequences analysed may be either the genomic DNA, thecDNA or the mRNA.

[0302] It will also be possible to use nucleic acids or antibodies basedon the present invention in order to allow a positive and differentialdiagnosis in a patient taken in isolation. The nucleic sequences will bepreferably used for a pre-symptomatic diagnosis in an at risk subject,in particular with a familial history. It is also possible to envisagean ante-natal diagnosis.

[0303] In addition, the detection of a specific mutation may allow anevolutive diagnosis, in particular as regards the intensity of thepathology or the probable period of its appearance.

[0304] The methods allowing the detection of a mutation in a genecompared with the natural gene are, of course, highly numerous. They canessentially be divided into two large categories The first type ofmethod is that in which the presence of a mutation is detected bycomparing the mutated sequence with the corresponding nonmutated naturalsequence, and the second type is that in which the presence of themutation is detected indirectly, for example by evidence of themismatches due to the presence of the mutation.

[0305] These methods can use the probes and primers of the presentinvention which are described. They are generally purified nucleicsequences for hybridization comprising at least 8 nucleotides,characterized in that they can hybridize specifically with a nucleicsequence chosen from the group comprising SEQ ID 1, SEQ ID 3, SEQ ID 5,SEQ ID 7, SEQ ID 9, SEQ ID 11, SEQ ID 13, SEQ ID 14 SEQ ID 15, SEQ ID 19and SEQ ID 41. Preferably, the specific hybridization conditions arelike those defined in the examples, or such that they ensure at least95% homology. The length of these nucleic sequences for hybridizationcan vary from 8, 10, 15, 20 or 30 to 200 nucleotides, particularly from20 to 50 nucleotides, more particularly from 20 to 30 nucleotides.

[0306] Among the methods for the determination of an allelicvariability, a mutation, a deletion, a loss of heterozygocity or agenetic abnormality, the methods comprising at least one stage for theso-called PCR (polymerase chain reaction) or PCR-like amplification ofthe target sequence according to the invention likely to exhibit anabnormality with the aid of a pair of primers of nucleotide sequencesaccording to the invention are preferred. The amplified products may betreated with the aid of an appropriate restriction enzyme beforecarrying out the detection or assay of the targeted product.

[0307] PCR-like will be understood to mean all methods using direct orindirect reproductions of nucleic acid sequences, or alternatively inwhich the labelling systems have been amplified, these techniques are ofcourse known, in general they involve the amplification of DNA by apolymerase; when the original sample is an RNA, it is advisable to carryout a reverse transcription beforehand. There are currently a greatnumber of methods allowing this amplification, for example the so-calledNASBA “Nucleic Acid Sequence Based Amplification” (Compton 1991), TAS“Transcription based Amplification System” (Guatelli et al., 1990), LCR“Ligase Chain Reaction” (Landegren et al., 1988), “Endo RunAmplification” (ERA), “Cycling Probe Reaction” (CPR), and SDA “StrandDisplacement Amplification” (Walker et al., 1992), methods well known topersons skilled in the art.

[0308] The invention comprises, in addition, methods for the diagnosisof pathologies and/or pathogeneses correlated with abnormal expressionof a polypeptide and/or a receptor according to the invention,characterized in that an antibody according to the invention is broughtinto contact with the biological material to be tested, under conditionsallowing the possible formation of specific immunological complexesbetween the said polypeptide and the said antibody, and in that theimmunological complexes possibly formed are detected.

[0309] Mutations in one or more genes of the LSR complex may beresponsible for various modifications of their product(s), whichmodifications can be used for a diagnostic approach. Indeed,modifications of antigenicity can allow the development of specificantibodies. The discrimination between the various conformations of LSRcan be achieved by these methods. All these modifications may be used ina diagnostic approach by virtue of several well-known methods based onthe use of mono- or polyclonal antibodies recognizing the normalpolypeptide or mutated variants, such as for example using RIA or ELISA.

[0310] These diagnostic methods also relate to the methods of diagnosisby imaging in vivo or ex vivo using the monoclonal or polyclonalantibodies according to the invention, particularly those labelled andcorresponding to all or part of the mutated polypeptides (imaging withthe aid of antibodies coupled to a molecule which is detectable inPET-scan type imaging, for example).

[0311] Screening of Compounds of Interest

[0312] Also included in the invention are the methods for selecting thechemical or biochemical compound capable of interacting, directly orindirectly, with the receptor according to the invention, and/orallowing the expression or the activity of the said receptor to bemodulated, characterized in that they use a receptor, a nucleic acid, apolypeptide, a vector, a cell or a mammal according to the invention.

[0313] Screening of Compounds Modifying the Activity of the LSR Receptor

[0314] The invention relates to a method for screening compoundsmodifying the activity of the LSR receptor, consisting in measuring theeffect of candidate compounds on various parameters reflecting, directlyor indirectly, taken independently or in combination, an LSR receptoractivity.

[0315] For the screening of compounds capable of modulating the LSRactivity for lipoprotein clearance, the preferred principal effect isthe effect of the compound on the activity of binding, internalizationand degradation of the lipoproteins by the LSR receptor.

[0316] This effect can be analysed in the absence or in the presence offree fatty acids, or of any other agent known to induce or to inhibitthe activity of LSR on the clearance of lipoproteins, or in the absenceor the presence of leptin, or of any other agent capable of inducing orof inhibiting the LSR function of cytokine clearance. It can, inaddition, be measured in the absence or in the presence of agentscapable of promoting or reducing the lipase activities, eitherintracellular or extracellular, as well as in the presence or in theabsence of alternative known routes of degradation of lipoproteins.

[0317] Various indirect parameters can also be measured, including thefollowing

[0318] the change in weight induced by the administration of thecompound

[0319] the food intake induced by the administration of the compound

[0320] the postprandial lipemic response induced by the administrationof the compound, before, during or after ingestion of a meal, forexample high in fat.

[0321] The selection of compounds capable of influencing the plasmatriglyceride concentrations, and/or the binding, internalization andhepatic degradation of lipoproteins or particles high in triglycerides,will be preferred.

[0322] For the screening of compounds capable of modulating the LSRactivity of clearance of cytokines, in particular of leptin, thepreferred principal effect is the effect of the compound on the activityof binding, internalization and hepatic degradation of cytokines by theLSR receptor, in the absence or in the presence of free fatty acids.

[0323] The measurement of the binding, internalization and/ordegradation of lipids or of cytokines can be carried out, for example,on hepatocytes or fibroblasts in culture, or on any other cellexpressing the LSR receptor at its surface. The cells will be preferablycells expressing a recombinant LSR receptor, more particularly cellsexpressing a recombinant LSR receptor and whose endogenous LSR receptorwould be inactivated or absent. These cells may or may not express theLDL receptor.

[0324] The screening of compounds modulating the LSR activity preferablyuses cells or model animals according to the invention, in particularmice, rats or humans, more particularly those described above and in theexamples which follow.

[0325] Screening of Compounds Modifying the Expression of the LSRReceptor

[0326] Screening may be used to test compounds capable of modifying thelevel and/or the specificity of expression of the LSR receptor either bybinding competitively to the sites for binding of transcription factorssituated in the LSR promoter or by binding directly to the transcriptionfactors.

[0327] The level of expression of the LSR receptor and its location canbe analysed by hybridization in solution with large probes as indicatedin Patent PCT WO 97/05277, the teaching of this document beingincorporated by reference. Briefly, a cDNA or the genomic DNA for theLSR receptor or alternatively a fragment thereof is inserted at acloning site situated directly downstream of a bacteriophage (T3, T7 orSP6) RNA polymerase promoter in order to produce an antisense RNA.Preferably, the insert comprises at least 100 consecutive nucleotides ofthe genomic sequence of the LSR receptor or of one of the cDNAs of thepresent invention, more particularly one or more of the cDNAs of SEQ ID9, SEQ ID 11 or SEQ ID 13. The plasmid is linearized and transcribed inthe presence of ribonucleotides comprising modified ribonucleotides suchas Biotin-UTP and Digoxigenin-UTP. An excess of this labelled RNA ishybridized in solution with the mRNAs isolated from cells or fromtissues of interest. The hybridizations are carried out under stringentconditions (40-50° C. for 16 h in a solution containing 80% formamideand 0.4 M NaCl, pH 7-8). The non-hybridized probe is eliminated bydigestion with ribonucleases specific for single-stranded RNAs (CL3, T1,PhyM, U2 or A RNases). The presence of modified nucleotides biotin-UTPallows the capture of the hybrids on microtitre plates carryingstreptavidine. The presence of the DIG modification allows the detectionand quantification of the hybrids by ELISA using anti-DIG antibodiescoupled to alkaline phosphatase.

[0328] A quantitative analysis of the expression of the gene for the LSRreceptor can also be carried out using DNA templates, the term DNAtemplates designating a one-dimensional, two-dimensional ormulti-dimensional arrangement of a plurality of nucleic acids having asufficient length to allow a specific detection of the expression ofmRNAs capable of hybridizing thereto. For example, the DNA templates maycontain a plurality of nucleic acids derived from genes for which it isdesired to estimate the level of expression. The DNA templates mayinclude the genomic sequences of LSR, that of a cDNA of the presentinvention, more particuliarly one or more of the cDNAs of SEQ ID 9, SEQID 11 or SEQ ID 13, any sequences complementary thereto or any fragmentsthereof. Preferably, the fragments comprise at least 15, at least 25, atleast 50, at least 100 or at least 500 consecutive nucleotides of thenucleic sequences from which they are derived.

[0329] For example, a quantitative analysis of the expression of the LSRreceptor can be carried out with a DNA template having the cDNA for theLSR receptor as described in Schena et al. (1995 and 1996). cDNAs forthe LSR receptor or fragments thereof are amplified by PCR and bound inthe form of a template from a 96-well microplate onto a sylatedmicroscope slide using a very fast automated machine. The DNA templatethus produced is incubated in a humid chamber in order to allow itsrehydratation. It is then rinsed once in 0.2% SDS for 1 min, twice inwater for 1 min and once for 5 min in a sodium borohydride solution. Thetemplate is then submerged in water for 2 min at 95° C., transferredinto 0.2% SDS for 1 min, rinsed twice with water, dried and stored inthe dark at 25° C.

[0330] The mRNAs of cells and of tissues are isolated or obtained from acommercial source, for example the company Clontech. The probes areprepared by a reverse transcription cycle. The probes are thenhybridized with the DNA template of 1 cm² under a glass coverslip of14×14 mm for 6-12 hours at 60° C. The template is washed for 5 min at25° C. in a washing buffer at low stringency (1×SSC/0.2% SDS) and thenfor 10 min at room temperature in a highly stringent buffer(0.1×SSC/0.2% SDS). The template is analysed in 0.1×SSC using a laserfluorescence microscope with a set of appropriate filters. Measurementsof precise differential expression are obtained by taking the mean ofthe ratios of two independent hybridizations.

[0331] A quantitative analysis of the expression of the LSR receptor canalso be carried out with cDNAs for the LSR receptor or fragments thereofon DNA templates according to the description by Pietu et al. (1996).The cDNAs for the LSR receptor or fragments thereof are amplified by PCRand bound to membranes. The mRNAs obtained from different tissues orcells are labelled with radioactive nucleotides. After hybridization andwashing under controlled conditions, the hybridized mRNAs are detectedwith a Phosphor Imager or by autoradiography. The experiments arecarried out in duplicate and a quantitative analysis of thedifferentially expressed mRNAs can be carried out.

[0332] Alternatively, the analysis of the expression of the LSR receptorcan be made with DNA templates at high density as described by Lockhartet al. (1996) and Sosnowski et al. (1997). Oligonucleotides of 15 to 50nucleotides, preferably about 20 nucleotides, extracted from genomic DNAor cDNA sequences for the LSR receptor or of their complementarysequences are synthesized directly on a chip or synthesized and thenaddressed onto the chip.

[0333] LSR cDNA probes labelled with an appropriate compound such asbiotin, digoxigenin or a fluorescent molecule are synthesized from apopulation of mRNA and are fragmented into oligonucleotides of 50 to 100nucleotides on average. The probes thus obtained are then hybridized toa chip. After washing as described in Lockhart et al (1996) and anapplication of various electric fields (Sosnowski et al. 1997), thelabelled compounds are detected and quantified. The hybridizations areduplicated. A comparative analysis of the intensity of the signalsgenerated by the probes on the same target oligonucleotide in variouscDNA samples indicates a differential expression of the mRNAs for theLSR receptor.

[0334] The techniques mentioned above allow the analysis of the levelsof expression of the LSR receptor, in the same cell or the same tissuedepending on various conditions, for example of induction or ofnoninduction, but also the analysis of the tissue specificity of thisexpression, under conditions which can also vary. It will be possible,by virtue of these techniques, to analyse the expression of either ofthe subunits of the LSR receptor, and more generally of different formsderived from alternative splicing, by adequately defining the probes.

[0335] The effect of compounds which are candidates for modulating thelevel or the specificity of expression, or of splicing of the differentforms of the LSR receptor can thus be analysed on a large scale byexposing the cells which are the source of messenger RNA, in particularthe model cells according to the invention, whether they express LSRnaturally or whether they are recombinant cells, to the said candidatecompounds.

[0336] Screening of Compounds Interacting With the LSR Receptor

[0337] Another aspect of the present invention consists in methods ofidentifying molecules capable of binding to the LSR receptor. Suchmolecules can be used to modulate the activity of the LSR receptor. Forexample, such molecules can be used to stimulate or reduce thedegradation of lipoproteins, preferably of lipoproteins high intriglycerides, or of cytokines, preferably of leptin. Such molecules canalso be used to inhibit the activation by leptin or the activation byfree fatty acids of the LSR activity.

[0338] Numerous methods exist for identifying ligands for the LSRreceptor. One of these methods is described in patent U.S. Pat. No.5,270,170, whose teaching is incorporated by reference. Briefly, alibrary is constructed which consists of random peptides, comprising aplurality of vectors each encoding a fusion between a peptide which is acandidate for binding to the LSR receptor and a protein binding to DNAsuch as the Lac repressor encoded by the lad gene. The vectors for thelibrary of random peptides also contain binding sites for the proteinsbinding to DNA such as the LacO site when the protein is the Lacrepressor. The library of random peptides is introduced into a host cellin which the fusion protein is expressed. The host cell is then lysedunder conditions allowing the binding of the fusion protein to the sitesof the vector.

[0339] The vectors which have bound the fusion protein are brought intocontact with the immobilized LSR receptor, a subunit of the immobilizedLSR receptor or a fragment of the immobilized LSR receptor underconditions allowing the peptides to bind specifically. For example, theLSR receptor, a subunit thereof or a fragment thereof comprising atleast 10, at least 20, at least 30, or more than 30 consecutive aminoacids can be immobilized by binding to a surface such as a plate or aplastic particle.

[0340] The vectors which encode the peptides capable of binding to theLSR receptor are specifically retained at the surface by interactionsbetween the peptide and the LSR receptor, a subunit of the receptor or afragment thereof.

[0341] Alternatively, molecules capable of interacting with the LSRreceptor can be identified using a double hybrid system such as theMatchmaker Two Hybrid System 2. According to the instructions of themanual accompanying the Matchmaker Two Hybrid System 2 (Catalogue No.K1604-1, Clontech), whose teaching is incorporated by reference, thenucleic acids encoding the LSR receptor, a subunit thereof or a fragmentthereof comprising at least 10, at least 20, at least 30, or more than30 consecutive amino acids are inserted into an expression vector sothat they are in phase with the DNA encoding the DNA binding domain ofthe transcription activator of yeast GAL4. The nucleic acids of alibrary encoding proteins or peptides capable of interacting with theLSR receptor are inserted into a second expression vector so that theyare in phase with the DNA encoding the activation domain of the GAL4activator. The yeasts are transformed with the two expression plasmidsand they are placed in a medium which makes it possible to select thecells expressing markers contained in each of the vectors as well asthose expressing the HIS3 gene whose expression is dependent on GAL4.The transformed cells capable of growing on a histidine-free medium areanalysed for expression of LacZ under the dependence of GAL4. The cellswhich grow in the absence of histidine and express LacZ contain aplasmid which encodes proteins or peptides which interact with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidsthereof.

[0342] To study the interaction of the LSR receptor, a subunit thereofor a fragment thereof comprising at least 10, at least 20, at least 30,or more than 30 consecutive amino acids with small molecules such asthose generated by combinatory chemistry, it is possible to use anHPLC-coupled microdialysis as described in Wang et al. (1997), or anaffinity capillary electrophoresis as described in Busch et al. (1997),the teaching of these documents being incorporated by reference.

[0343] In other methods, the peptides or small molecules capable ofinteracting with the LSR receptor, a subunit thereof or a fragmentthereof comprising at least 10, at least 20, at least 30, or more than30 consecutive amino acids may be linked to detectable markers such asradioactive, fluorescent or enzymatic markers. These labelled moleculesare brought into contact with the immobilized LSR receptor, animmobilized subunit thereof or an immobilized fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids under conditions allowing a specificinteraction. After elimination of the molecules which are notspecifically bound, the bound molecules are detected by appropriatemeans.

[0344] These methods may allow in particular the identification of fattyacids or analogues capable of binding to the fatty acid binding site onthe LSR, of lipoproteins or analogues, capable of binding to thelipoprotein binding site on the LSR receptor, of leptin derivatives oranalogues capable of binding to the leptin binding site on the LSR, andof derivatives of the gC1qR receptor or analogues capable of binding tothe gC1qR binding site on the LSR.

[0345] In addition, the peptides or small molecules which bind to LSR,preferably to the binding sites on the LSR receptor for fatty acids,lipoproteins, cytokines, in particular leptin, or gC1qR or one of itsanalogous proteins, can be identified by competition experiments. Insuch experiments, the LSR receptor, a subunit thereof or a fragmentthereof comprising at least 10, at least 20, at least 30, or more than30 consecutive amino acids is immobilized on a surface such as a plasticsupport. Increasing quantities of peptides or of small molecules arebrought into contact with the immobilized LSR receptor, an immobilizedsubunit thereof or an immobilized fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acids inthe presence of a labelled ligand for the receptor, it being possiblefor this ligand to be, for example, leptin, oleate, the LDLs or gC1qR.The ligand for the LSR receptor may be labelled with a radioactive,fluorescent or enzymatic marker. The capacity of the molecule tested tointeract with the LSR receptor, a subunit thereof or a fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids is determined by measuring the quantity oflabelled ligand bound in the presence of the molecule tested. A decreasein the quantity of bound ligand when the molecule tested is presentindicates that the latter is capable of interacting with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acids.

[0346] These methods can in particular allow the identification of fattyacids or analogues capable of binding to the fatty acid binding site onthe LSR, of lipoproteins or analogues, capable of binding to thelipoprotein binding site on the LSR receptor, of leptin derivatives oranalogues capable of binding to the leptin binding site on the LSR, andof derivatives of the gC1qR receptor or analogues capable of binding tothe gC1qR binding site on the LSR. The capacity of such compounds, or ofany other candidate compound, to compete with the binding of oleates,lipoproteins, leptin or gC1qR to LSR can be measured in particular.

[0347] The BIACORE technology can also be used to carry out thescreening of compounds capable of interacting with the LSR receptor.This technology is described in Szabo et al. (1995) and in Edwards andLeartherbarrow (1997), of which the teaching is incorporated byreference, and makes it possible to detect interactions betweenmolecules in real time without the use of labelling. It is based on thephenomenon of SPR (surface plasmon resonance). Briefly, the molecule tobe analysed is bound to a surface (typically using a carboxymethyldextran matrix). A light ray is directed onto the face of the surfacewhich does not contain the sample and is reflected by the said surface.The SPR phenomenon causes a reduction in the intensity of the reflectedlight with a specific combination of angle and of wavelength. Themolecule binding events cause a change in the refractive index at thesurface which is detected as a modification of the SPR signal. To carryout a screening of compounds capable of interacting with the LSRreceptor, the LSR receptor, a subunit thereof or a fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids, is immobilized on a surface. This surfaceconstitutes one face of a cell through which passes the molecule to betested. The binding of the molecule to the LSR receptor, a subunitthereof or a fragment thereof comprising at least 10, at least 20, atleast 30, or more than 30 consecutive amino acids is detected by achange in the SPR signal. The molecules tested may be proteins,peptides, carbohydrates, lipids or small molecules generated, forexample, by combinatory chemistry. The candidate proteins can beextracted from any tissue, obtained from any species. The BIACOREtechnology can also be used by immobilizing eukaryotic or prokaryoticcells or lipid vesicles having an endogenous or recombinant LSR receptorat their surface.

[0348] One of the main advantages of this method is that it allows thedetermination of the association constants between the LSR receptor andthe interacting molecules. Thus, it is possible to specifically selectthe molecules interacting with high or low association constants.

[0349] The proteins or other molecules interacting with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidscan be identified using affinity columns which contain the LSR receptor,a subunit thereof or a fragment thereof comprising at least 10, at least20, at least 30, or more than 30 consecutive amino acids. The LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidsmay be attached to the column using conventional techniques includingchemical coupling to an appropriate column matrix such as agarose, AffiGel, or other matrices known to a person skilled in the art. In anotheraspect of the invention, the affinity column may contain chimericproteins in which the LSR receptor, a subunit thereof or a fragmentthereof comprising at least 10, at least 20, at least 30, or more than30 consecutive amino acids would be fused, for example, with glutathioneS-transferase. The molecules to be tested which are described above arethen deposited on the column. The molecules interacting with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidsare retained by the column and can be isolated by elution. In the casewhere the molecules tested are proteins, they can then be analysed on a2-D electrophoresis gel as described in Ramunsen et al. (1997), of whichthe teaching is incorporated by reference. Alternatively, the proteinsor the other molecules retained by the affinity column can be purifiedby electrophoresis and sequenced. A similar method can be used toisolate antibodies, to screen “phage display”, products or “phagedisplay ” derived human antibodies.

[0350] Screening of Compounds Interacting With the Promoter and/orRegulatory Sequences of the LSR Receptor

[0351] The invention also relates to a method of screening compoundsinteracting with the promoter and/or regulatory sequences of the LSRreceptor.

[0352] The nucleic acids encoding proteins interacting with the promoterand/or regulatory sequences of the LSR receptor gene, more particularlya nucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID19 or a fragment thereof, can be identified using a single hybrid systemsuch as that described in the manual accompanying the MatchmakerOne-Hybrid System from Clontech (Catalogue No. K1603-1), of which theteaching is incorporated by reference. Briefly, the target nucleotidesequence is cloned upstream of a selectable marker gene and integratedinto a yeast genome. The yeasts containing the integrated marker geneare transformed by a library containing fusions between cDNAs encodingcandidate proteins for binding to the promoter and/or regulatory regionsof the gene for the LSR receptor and the yeast transcription factoractivating domain such as GAL4. The yeasts are placed in a medium whichmakes it possible to select the cells expressing the marker gene. Theyeasts selected contain a fusion protein capable of binding to thepromoter and/or regulatory target region. The cDNAs of the genesencoding the fusion proteins are then sequenced. The correspondinginserts can then be cloned into expression or transcription vectors invitro. The binding of the polypeptides thus encoded to the promotertarget sequences can be confirmed by techniques familiar to personsskilled in the art, including gel retardation or protection to DNAseexperiments.

[0353] The screening of compounds capable of modifying the expression ofthe LSR receptor by binding to its regulatory and/or promoter sequencescan also be carried out with the aid of “reporter” genes. For example, agenomic region situated in 5′ of the coding sequence of the LSRreceptor, more particularly a nucleotide sequence corresponding tonucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, can be clonedinto a vector such as pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic,pβgal-Enhancer, or pEGFP-1 available from Clontech. Briefly, each ofthese vectors contains multiple cloning sites situated upstream of amarker gene encoding an easily detectable protein such as alkalinephosphatase, β-galactosidase or GFP (green fluorescent protein). Afterinsertion of the genomic region situated in 5′ of the coding sequence ofthe LSR receptor, more particularly a nucleotide sequence correspondingto nucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, the levelof expression of the marker proteins is measured and compared with avector containing no insert. The effect of candidate compounds on theexpression resulting from the regulatory and/or promoter sequences ofLSR can thus be evaluated.

[0354] The screening of the compounds capable of binding to theregulatory and/or promoter regions of the gene for the LSR receptor canalso be carried out by gel retardation experiments well known to personsskilled in the art and described in Fried and Crothers (1981), Garnerand Revzin (1981) and Dent and Latchman (1993), of which the teaching isincorporated by reference. These experiments are based on the principlethat a DNA fragment bound to a protein migrates more slowly than thesame fragment without protein. Briefly, the target nucleotide sequenceis labelled. It is then brought into contact either with a nuclear ortotal cell extract prepared so as to contain the transcription factors,or with various compounds to be tested. The interaction between theregulatory and/or promoter region of the gene for the LSR receptor andthe transcription factor or compound is detected after electrophoresisby retardation of migration.

[0355] Compounds

[0356] The chemical or biochemical compounds, characterized in that theymake it possible to modulate the expression or the activity of thereceptor according to the invention, also form part of the invention.

[0357] The chemical or biochemical compounds, characterized in that theyare capable of interacting, directly or indirectly, with the receptoraccording to the invention, also form part of the invention.

[0358] The chemical or biochemical compounds, characterized in that theyare selected by the said methods defined above, also form part of theinvention.

[0359] In particular, among these compounds according to the invention,a leptin or one of its derived compounds, preferably one of its proteinvariants, or leptins which are chemically modified or which are obtainedby genetic recombination, or one of their fragments, are preferred.

[0360] Compounds which make it possible to modulate the expression orthe activity of the receptor are understood to mean the compounds whichmake it possible in particular to reduce, stabilize or increase thenumber, the recycling rate and/or the change in the conformation of thereceptor according to the invention, or to promote or inhibit theoverall activity or the activity of one of the domains of the saidreceptor or alternatively to reestablish normal expression of the saidreceptor in the case, for example, where a genetic abnormality isobserved. These compounds may, for example, interact as ligands specificfor the said receptor or for one of its domains as cofactor, or asinhibitor, in particular a competitive inhibitor, or alternativelyhaving an agonist or antagonist activity on the conformational changesin the complex. These compounds may also interact by neutralizing thenatural ligands specific for the said receptor and by thereby inhibitingthe receptor activity induced by these ligands.

[0361] Among these compounds, the compounds which make it possible tomodulate the number of polypeptides of the said receptor, its recyclingrate and/or the selectivity of their activity, are preferred.

[0362] Also preferred are the compounds according to the invention,characterized in that they allow an increase in the total activity or inthe expression of the receptor according to the invention, and/or aspecific increase in the clearance activity for cytokines, in particularleptin, of the said receptor, and/or a specific increase in theclearance activity for lipoproteins, of the said receptor.

[0363] Also preferred are the compounds characterized in that they allowa decrease in the total activity or in the expression of the receptoraccording to the invention, and/or a specific decrease in the clearanceactivity for cytokines, in particular leptin, of the said receptor,and/or a specific decrease in the clearance activity for lipoproteins,of the said receptor.

[0364] Also preferred are the compounds characterized in that they allowmodulation of the elimination of the cytokines, in particular leptin,and/or modulation of the elimination of the lipoproteins, chylomicronresidues, and/or triglycerides.

[0365] The invention also comprises the compounds according to theinvention, characterized in that they allow modulation of the level ofcytokines, in particular leptinemia, and/or modulation of the level oflipoproteins, chylomicron residues, and/or triglycerides.

[0366] The compounds according to the invention, characterized in thatthey allow control of the level of cytokines, in particular leptinemia,are more particularly preferred.

[0367] Still preferably, the invention comprises the compounds accordingto the invention, characterized in that they allow control, preferably adecrease, of the level of lipoproteins, a decrease in the plasmaconcentration of chylomicron residues, and/or a decrease intriglyceridernia.

[0368] Among the compounds which are most preferred, there are preferredthose characterized in that they are chosen from:

[0369] a. an antibody according to the invention;

[0370] b. a polypeptide according to the invention;

[0371] c. a polypeptide according to the invention, characterized inthat it corresponds to a soluble form of the receptor according to theinvention;

[0372] d. a vector according to the invention;

[0373] e. a vector according to the invention, characterized in that ithas on its outer surface a site for specific recognition of hepaticcells;

[0374] f. a vector according to the invention, characterized in that theproduct of expression of the nucleic acid inserted by the vector intothe target cell is either anchored in or excreted by the saidtransformed target cell;

[0375] 9. a sense or antisense oligonucleotide according to theinvention;

[0376] h. a leptin, or one of its protein variants, or a leptin which ischemically modified or which is modified by genetic recombination, orone of their fragments.

[0377] The invention finally relates to the compounds according to theinvention as a medicament.

[0378] The compounds according to the invention as a medicament for theprevention and/or treatment of pathologies and/or of pathogeneses linkedto disorders in dietary habit are preferred in particular.

[0379] The compounds according to the invention as a medicament for theprevention and/or treatment of pathologies and/or of pathogeneses linkedto disorders in the metabolism of cytokines are also preferred.

[0380] Preferably, the invention also relates to the compounds accordingto the invention as medicament for the prevention or treatment ofobesity or anorexia.

[0381] The compounds according to the invention as a medicament for theprevention and/or treatment of pathologies and/or of pathogenesesassociated with, or induced by obesity, are the preferred compounds.

[0382] In particular, there are preferred the compounds according to theinvention, as a medicament for the prevention and/or treatment ofcardiac insufficiency, of coronary insufficiency, of cerebrovascularaccidents, of atheromatous disease, of atherosclerosis, of high bloodpressure, of non-insulin-dependent diabetes, of hyperlipidemia and/or ofhyperuricemia.

[0383] The most preferred are the compounds according to the invention,as a medicament for the prevention and/or treatment of atheromatousdisease and/or of atherosclerosis.

[0384] Finally, the invention comprises compounds according to theinvention for the prevention and/or treatment by gene therapy, ofpathologies and/or of pathogeneses linked to disorders in dietary habit,of obesity and/or of pathologies and/or of pathogeneses associated with,or induced by, obesity.

[0385] The compounds of the invention as active ingredients of amedicament will be preferably in soluble form, combined with apharmaceutically acceptable vehicle.

[0386] Such compounds which can be used as a medicament offer a newapproach for preventing and/or treating pathologies and/or pathogeneseslinked to disorders in dietary habit such as obesity or anorexia, andthe related risks and/or complications.

[0387] Preferably, these compounds will be administered by the systemicroute, in particular by the intravenous route, by the intramuscular orintradermal route or by the oral route.

[0388] Their modes of administration, optimum dosages and galenic formscan be determined according to the criteria generally taken into accountin establishing a treatment suited to a patient, such as for example theage or body weight of the patient, the seriousness of his generalcondition, the tolerance to treatment and the side effects observed, andthe like.

[0389] As mentioned above, depending on the cases, it may be advisableto amplify the activity of LSR, by promoting, for example, theexpression of its genes or by increasing the activity of theirexpression products, in pathological cases resulting from the fact thatat least one of these genes is not expressed, is insufficientlyexpressed or is expressed in an abnormal form which does not allow theexpression product to carry out its functions, or on the contrary torepress an overexpression or an abnormal expression of these genes. Itis therefore advisable in general to compensate for the deficiency orthe overexpression of expression products of this gene by a so-called“replacement” therapy allowing the amplification or the reduction in theactivities of the LSR complex.

[0390] The replacement therapy may be carried out by gene therapy, thatis to say by introducing the nucleic acid sequences according to theinvention and/or the corresponding genes with the elements which allowtheir expression in vivo, in the case where one of the genes isinsufficiently expressed for example, or alternatively when it isexpressed in an abnormal form.

[0391] The principles of gene therapy are known. It is possible to useviral vectors according to the invention; it is also possible toenvisage nonviral, that is to say synthetic, vectors which mimic viralsequences or alternatively which consist of naked RNA or DNA accordingto the technique developed in particular by the company VICAL.

[0392] In most cases, it is necessary to envisage targeting elementsensuring expression specific for the liver so as to be able to limit thezones of expression of the proteins which remain involved in theclearance of leptin and that of lipoproteins. It is even advantageous,in some cases, to have vectors for transient expression or at least forcontrolled expression which it will be possible to block when necessary.

[0393] Other characteristics and advantages of the invention appear inthe remainder of the description with the examples and figures whoselegends are represented below.

LEGEND TO THE FIGURES

[0394]FIG. 1: Schematic representation of the three forms of the rat LSRprotein: LSR 66 (α subunit), LSR 64 (α′ subunit), and LSR 58 (αsubunit).

[0395]FIG. 2: Alignment of the protein sequences of the long forms (αsubunits) of the human LSR (LSR1.Hs; SEQ ID NO:8), rat LSR (LSR1.Rn; SEQID NO:2) and mouse LSR (LSR1.Mm; SEQ ID NO:16). The (*) symbols placedunder the alignments indicate the conserved amino acids, the (.) symbolsindicate the conservative substitutions of amino acids. Boxed, from theNH₂-terminal end to the COOH-terminal end, the potential fatty acid(FFA) binding site boxed, the clathrin binding site [NPGY], the lyosomaladdressing consensus: di-leucine LI-X10-LL, the transmembrane TM domainoverlined, the motif [RSRS], the potential lipoprotein binding site(+-+-) boxed. Overlined, the signature of the TNF receptor with (arrow);indicated, the amino acids conserved in the signature. The transmembranedomain is situated between the last di-leucine and the TNF signature.

[0396] A: Alignment shown from amino acid positions 1 to 539 of SEQ IDNO:8.

[0397] B: Alignment shown from amino acid positions 540 to 649 of SEQ IDNO:8.

[0398]FIG. 3: Alignment of the protein sequences of the three types ofsubunits of the human LSR (α: LSR1.Hs, SEQ ID NO:8; α′: LSR2.Hs, SEQ IDNO:10; P: LSR3.Hs, SEQ ID NO:12). The meaning of the symbols, of theboxes and of the overlines is the same as that in FIGS. 2A and 2B.

[0399] A: Alignment shown from amino acid positions 1 to 540 of SEQ IDNO:8.

[0400] B: Alignment shown from amino acid positions 541 to 649 of SEQ IDNO:8.

[0401]FIG. 4: Alignment of the protein sequences of the three types ofsubunits of rat LSR. (α: LSR1.Rn, SEQ ID NO:2; α′: LSR2.Rn, SEQ ID NO:4;β: LSR3.Rn, SEQ ID NO:6). The meaning of the symbols, of the boxes andof the overlines is the same as that in FIGS. 2A and 2B.

[0402] A: Alignment shown from amino acid positions 1 to 540 of SEQ IDNO:2.

[0403] B: Alignment shown from amino acid positions 541 to 593 of SEQ IDNO:2.

[0404]FIG. 5: Alignment of the protein sequences of the three types ofsubunits of mouse LSR (a: LSR1.Mm, SEQ ID NO:16; α′: LSR2.Mm, SEQ IDNO:17; α′: LSR3.Mm, SEQ ID NO:18). The meaning of the symbols, of theboxes and of the overlines is the same as that in FIGS. 2A and 2B.

[0405] A: Alignment shown from amino acid positions 1 to 540 of SEQ IDNO:16.

[0406] B: Alignment shown from amino acid positions 541 to 594 of SEQ IDNO:16.

[0407]FIG. 6: Schematic representation of the three LSR forms identifiedin humans, indicating the motifs conserved on each of them.

[0408] A: Schematic representation of the genomic organization of thehuman LSR starting from the first coding exon. The exons are indicatedby boxes, the introns by interrupted bars. The size, in nucleotides, ofthe exons and introns is indicated above them. The elementscharacterizing the messenger and the encoded protein are presented inthis figure. The box on the right gives the meaning of the symbols used.

[0409] B: Structure of the LSR-Hs-2062 form of human LSR. This formencodes a protein of 649 amino acids.

[0410] C: Structure of the LSR-Hs-2005 form of human LSR. This formencodes a protein of 630 amino acids.

[0411] D: Structure of the LSR-Hs-1858 form of human LSR. This formencodes a protein of 581 amino acids.

[0412]FIG. 7: Alignment of the nucleotide sequences of the long forms ofcDNA (encoding the α subunit) or portions thererof for human LSR(Isr1.HS; nucleotides 1 to 2062 of SEQ ID NO:7), rat LSR (Isr1.Rn; SEQID NO:1) and mouse LSR (Isr1.Mm; SEQ ID NO:13). The nucleotidesconserved in the three sequences are identified by an * sign placedunder the sequences. Dashes are added inside the sequences when theoptimum alignment of the sequences cannot be achieved without creatingmicrodeletions.

[0413] A: Alignment shown from amino acid positions 1 to 486 of SEQ IDNO:1.

[0414] B: Alignment shown from amino acid positions 487 to 1026 of SEQID NO:1.

[0415] C: Alignment shown from amino acid positions 1027 to 1551 of SEQID NO:1.

[0416] D: Alignment shown from amino acid positions 1552 to 2080 of SEQID NO:1.

[0417] E: Alignment shown from amino acid positions 2081 to 2097 of SEQID NO:1.

[0418]FIG. 8: Identification of the LSR receptor by ligand and Westernblotting on solubilized proteins of rat liver membranes (lanes 1, 2 and4), or on the partially purified protein of 240 kD (lane 3).

[0419] Lanes 1, 2 and 3: Ligand blotting. Lane 1: in the absence ofoleate and of ¹²⁵I-LDL; lane 2: in the presence of oleate and of¹²⁵I-LDL; lane 3: in the presence of oleate and of ¹²⁵I-LDL.

[0420] Lane 4: Western blotting with anti-LSR antibodies.

[0421]FIG. 9: Effect of anti-LSR antibodies on the LSR activity.

[0422] A. Binding of ¹²⁵I-LDL onto the plasma membranes of rathepatocytes in the presence of oleate and of increasing concentrationsof anti-LSR antibody (ν) or of control antibody (O), expressed as % ofthe total quantity of ¹²⁵I-LDL bound in the absence of antibodies.

[0423] B. Binding, incorporation and degradation of ¹²⁵I-LDL in rathepatocytes in primary culture in the presence of oleate and of anti-LSRantibody (ν) or of control antibody (O), expressed respectively as % ofthe binding, incorporation and total degradation of ¹²⁵I-LDL in thepresence of non-specific antibodies.

[0424]FIG. 10: Identification of the LSR receptor by immunoprecipitationof ³⁵S-methionine and ³⁵S-cysteine-labelled hepatocyte lysates, in thepresence of control antibodies (lane 1), or of anti-LSR antibodies(lanes 2 to 4), after separation by electrophoresis under nonreducing(lanes 2 and 3) or reducing (lanes 1 and 3) conditions.

[0425]FIG. 11: Cloning of the cDNA encoding α and β-LSR.

[0426] A. Northern-blot analysis showing several sizes of LSR messengerRNA.

[0427] B. Multi-tissue Northern-blot analysis of LSR mRNA with a probespecific for LSR and a control probe specific for β-actin.

[0428] C. RT-PCR analysis of LSR mRNA using 5 pairs of primers coveringthe entire sequence and identification of three forms derived fromalternative splicing in the amplification fragment obtained by means ofthe bc′ primers. The diagram represents the results of sequence analysisof the three corresponding forms of LSR cDNA: the squared region isabsent from the two short forms, the hatched region is absent only fromthe shortest form.

[0429]FIG. 12: Translation in vitro of the two complete cDNAs encodingthe longest (66 kDa, lane 2) and the shortest (58 kDa, lane 3) forms ofrat LSR, and of a control cDNA, an antisense of the cDNA encoding thelongest form of LSR (lane 1).

[0430] The products of translation in vitro, labelled with³⁵S-methionine, are analysed after electrophoresis under nonreducingconditions.

[0431]FIG. 13: Identification of the α and β-LSR subunits as beingresponsible for the LSR activity.

[0432] A. Diagram showing the location and the sequence of LSRN-terminal peptide used to generate anti-LSR peptide antibodies.

[0433] B. Effect of antibodies directed against a synthetic LSR peptideon the LSR activity of rat liver plasma membranes. The LSR activity ismeasured in the presence of a control antibody (O) or of the anti-LSRpeptide antibody (ν).

[0434] C. Western and Ligand blotting of the α and β subunits of LSR.The Western blotting is carried out using the anti-LSR (lane 1) oranti-LSR peptide (lane 2) antibody. The ligand blotting is carried outin the presence of ¹²⁵I-LDL, with (lane 4) or without (lane 3) oleate.

[0435]FIG. 14: Identification of the subunits of the LSR receptor andinhibitory effect of antibodies directed against a C-terminal syntheticpeptide derived from LSR.

[0436] A—Diagram showing the location and the sequence of the syntheticpeptide 170.

[0437] B—Western blotting of rat hepatocyte lysates using antibodiesdirected against the synthetic peptide 170 (lane 2), or a controlantibody (lane 1); lane 3: molecular weight markers.

[0438] C—Binding of ¹²⁵I-LDL by the LSR receptor in the presence ofoleate and of control antibodies or antibodies directed against the LSR170 peptide.

[0439]FIG. 15: Effect of a transient transfection of CHO-K1 cells withthe plasmids expressing the α and β subunits of the LSR receptor on thebinding of LDLs in the presence or in the absence of oleate. Increasingconcentration of α plasmid alone (◯□) fixed concentration of α plasmidand increasing concentration of β plasmid (▪).

[0440]FIG. 16: Effect of a transient transfection of CHO-K1 cells withplasmids expressing the a and β subunits of the LSR receptor on theinternalization and degradation of LDLs. Increasing concentration of αplasmid alone (▪); fixed concentration of α plasmid and increasingconcentration of β plasmid ( ). The results are expressed as thedifference between the measurements in the presence and in the absenceof oleate.

[0441]FIG. 17: Characterization of the LSR activity obtained in CHO-K1cells transiently transfected with the nucleic sequences encoding the αand β subunits of the LSR receptor, compared with the LSR activityobtained in the same cells not transfected (control).

[0442] A—Binding of ¹²⁵I-LDL in the presence of a control antibody or ofan anti-LSR antibody.

[0443] B—Binding of ¹²⁵I-LDL in the presence of increasingconcentrations of unlabelled lipoproteins; rat chylomicrons (υ), humanVLDL (▪), LDL (□), HDL (σ), LDLs treated with pronase (◯), or LDLsmodified with cyclohexanedione (LDL-chd, ).

[0444]FIG. 18: Effect of oleate, of RAP-39, of anti-LSR antibodies andof chloroquine on the specific degradation of leptin in primary culturesof rat hepatocytes.

[0445]FIG. 19: Western blot analysis with anti-LSR antibodies, of thefraction of rat liver plasma membrane proteins retained on an affinitychromatography column containing leptin.

[0446]FIG. 20: Clearance of ¹²⁵I-leptin on control (O), ob/ob (ν) anddb/db (z,1) mice in the liver and the kidney. The results are expressedas the difference between the quantities of ¹²⁵I-leptin and¹²⁵I-β2-microglobulin found in the liver and in the kidney.

[0447]FIG. 21: Apparent number of LSR receptors expressed in the liverof control, ob/ob and db/db mice.

[0448]FIG. 22: Effect of anti-LSR antibodies on the proportion betweenthe quantities of ¹²⁵I-leptin distributed in the liver and in thekidney.

[0449]FIG. 23: Effect of increasing leptin concentrations on the LSRactivity of rat hepatocytes in primary cultures. The results representthe differences in activity which are obtained between the cellsincubated with and without oleate in the presence either of ¹²⁵I-LDL, orof ¹²⁵I-VLDL.

[0450]FIG. 24: Capacity for inducing, by leptin, the LSR activity of rathepatocytes in primary culture.

[0451] A. Apparent number of receptors expressed at the surface of thehepatocytes in the presence or in the absence of leptin, estimated bythe measurement of the quantity of ¹²⁵I-LDL bound in the presence ofoleate.

[0452] B. Effect of cycloheximide, of colchicine and of cytochalasin Bon the induction, by leptin, of the LSR activity.

[0453]FIG. 25: Effect of leptin on the postprandial lipemic response incontrol (o), ob/ob (ν) and db/db (o) mice, reflected by the variation inthe plasma concentration of triglycerides (TG) after ingestion of ahigh-fat meal, with (B) and without (A) injection of murine recombinantleptin.

[0454]FIG. 26: Effect of leptin, in the presence and in the absence oflactoferrin, on the postprandial lipemic response of ob/ob mice,expressed by the measurement of the plasma concentration oftriglycerides (TG) after ingestion of a high-fat meal.

[0455]FIG. 27: Effect of leptin injection on the apparent number of LSRreceptors expressed in the liver of ob/ob and db/db mice.

[0456]FIG. 28: Postprandial lipemic response and LSR activity in control(C57BL6), ob/ob and db/db mice.

[0457] A—Weight of control, ob/ob and db/db male mice.

[0458] B—Postprandial lipemic response in control, ob/ob and db/db mice.

[0459] C—Apparent number of LSR receptors estimated by measurement ofthe binding of LDL and expressed in arbitrary unit by comparison withthe 5′-nucleotidase activity in each plasma membrane preparation.

[0460] D—Northern blot on an extract of liver total RNA. GAPDH is usedas control.

[0461]FIG. 29: Effect of a long-term treatment by leptin on ob/ob mice.

[0462] A—Weight change over 30 days

[0463] B—Postprandial lipemic response on the 29th day of treatment

[0464] C—Apparent number of LSR receptors on day 30, estimated by themeasurement of the binding of LDL and expressed in arbitrary unit bycomparison with the 5′-nucleotidase activity in each plasma membranepreparation

[0465] D—Northern blot analysis of the expression of LSR established ona total extract of liver RNA. GAPDH and actin are used as controls.

[0466]FIG. 30: Effect of the oleates on the binding and internalizationof the ¹²⁵I-LDL in normal human fibroblasts, under normal conditions.

[0467]FIG. 31: Effect of increasing concentrations of leptin on the LSRactivity of human fibroblasts HF (familial hypercholesterolemia).

[0468]FIG. 32: Inhibitory effect of antibodies directed against anNH₂-terminal (ν) or COOH-terminal (O) peptide of gC1qR, or of controlantibodies (o) on the LSR activity of plasma membranes of rathepatocytes, expressed as a percentage of the quantity of ¹²⁵I-LDL boundin the absence of antibodies.

[0469]FIG. 33: Effect of increasing concentrations of C1q on thebinding, internalization and degradation of ¹²⁵I-LDL on rat hepatocytesin primary culture, in the presence (ν) or in the absence (o) of oleate.

[0470]FIG. 34: Effect of 25 ng/ml of recombinant AdipoQ on the LSRactivity in a primary culture of rat hepatocytes.

[0471]FIG. 35: Effect of two successive injections of 1 mg of AdipoQ onthe postprandial lipemic response in rats after ingestion of a high-fatmeal.

[0472]FIG. 36: Effect of an intraperitoneal administration of AdipoQ for3 days on the weight and the concentrations of plasma triglycerides inrats on a normal diet or on a fatty diet.

[0473]FIG. 37: Effect of a daily injection of 100 μg of AdipoQ over 5days, on food intake in ob/ob and db/db obese mice.

EXAMPLES

[0474] Experimental Procedures

[0475] Materials

[0476] Na¹²⁵I is provided by Amersham (Les Ulis, France). Oleic acid,bovine serum albumin (A 2153) (BSA) and Triton X100 are obtained fromSigma (St Quentin Fallavier, France). Human lactoferrin (Serva) andsodium heparin are provided by Biowhittaker (Fontenay sous Bois, France)and Choay laboratories (Gentilly, France) respectively. The enzymatickits for the determination of triglycerides (TG) are obtained fromBoehringer Mannheim (Meylan, France). Suramin sodium is obtained fromCBC Chemicals (Woodburg, CT). Dulbecco's modified Eagle medium (DMEM),trypsin and foetal calf serum are provided by Life Technologies, Inc.(Eragny, France).

[0477] Animals

[0478] The mice C57BL/6J of the wild type, C57BL/6J ob/ob, C57BL/Ks ofthe wild type and C57BL/Ks db/db are obtained from R. Janvier BreedingCenter (Le Genest St Isle, France).

[0479] Cells

[0480] Normal fibroblasts (GM08333) and HF (GM00486A, GM007001B,GM00488C) are provided by the NIGMS human genetic mutant cell repository(Camden, NJ). The cells were plated on Petri dishes of 36 mm asdescribed above (300,000 normal fibroblasts per well, 150,000 HFfibroblasts per well), and are cultured in a humidified CO₂ incubator,in DMEM medium containing 10% (normal fibroblasts) or 20% (HFfibroblasts) foetal calf serum, 2 mM glutamine, 100 U/ml of penicillinand 100 U/ml of streptomycin.

[0481] The hepatocytes in primary culture are obtained according to theprocedure described above (Mann et al., 1995). The cells are then platedat 900,000 cells per well or 22×10⁶ cells per flask of 165 cm². Thecells are used for the studies after 48 hours in culture.

[0482] Preparation and Radiolabelling of the Lipoproteins

[0483] The VLDLs (d<1.006 g/ml) and LDLs (1.025<d<1.055 g/ml) areisolated by sequential ultracentrifugation of fresh plasma fromvolunteers (Bihain and Yen, 1992; Goldstein et al., 1983) and usedbefore 2 weeks. The lipoproteins are radioiodinated (Bilheimer et al.,1972) and used less than one week after the labelling. ¹²⁵I-LDL and¹²⁵I-VLDL are filtered (0.22 μm membrane, Gelman) immediately beforeuse.

[0484] Preparation and Radiolabelling of Mouse Recombinant Leptin

[0485] The leptin cDNA is obtained from the mRNA of adipose tissue ofthe mouse C57BL/6J by PCR. The 5′ PCR primer introduces an initiationcodon after the signal sequence which is deleted and a sequence encodinga hexahistidine end. The modified sequence encoding murine leptin iscloned into an expression vector pSE280 (Invitrogen, France) andexpressed in E. coli The sequencing of the plasmid DNA confirms thecoding sequence. The bacteria are cultured at 37° C. and the synthesisof the protein is induced by 1 mM isopropyl $-D-thiogalactopyranoside.The bacteria, recovered after gentle centrifugation, are lysed byfreeze-thaw and the DNA is digested with a deoxyribonuclease 1. Thecellular membranes are extracted with the aid of a detergent and theinclusion bodies are separated after centrifugation. After 3 washes in1% sodium deoxycholate in PBS, the inclusion bodies are solubilized in a6 M guanidine HCl solution. The renaturation of the recombinant proteinis achieved by diluting 1/100 in PBS. The renatured protein is thenpurified and concentrated on a nickel-based chelate metal affinitychromatography column (Probond, Invitrogen). The elution is carried outwith imidazole. The purity of the recombinant leptin is controlled bySDS-PAGE electrophoresis and its activity by the evaluation of satietyin mice C57BL/6J ob/ob after intraperitoneal injection of 25 μg ofleptin. The recombinant leptin is then radiolabelled using lodobeads(Pierce) and according to the method recommended by the manufacturer.

[0486] Cloning of the AdipoQ mRNA. Production and Purification ofRecombinant AdipoQ Proteins

[0487] Cloning of the cDNA into an expression vector

[0488] Mouse adipose tissue is obtained from C57BI/6J mice and the mRNAis extracted with the aid of polydTs bound to magnetic beads (Dynabeads,Dynal, France). A cDNA library is constructed from mouse adipose tissueby reverse transcription at 40° C. using a commercial kit (SuperscriptLife Technologies) using the supplier's instructions. The cDNA specificfor AdipoQ is amplified using the following two primers:

[0489] 5′CTACATGGATCCAGTCATGCCGAAGAT 3′ (SEQ ID 37)

[0490] 5′CGACAACTCGAGTCAGTTGGTATCATGG 3′ (SEQ ID 38).

[0491] The amplification product is then digested with the restrictionenzymes BamHI and XhoI and inserted into an expression vector pTRC HisB(Invitrogen, France) at the corresponding sites. The B version of pTRCallows the expression of heterogeneous sequences downstream of ahexahistidine peptide which carries a recognition site for anenterokinase and an epitope for the anti-Xpress antibody.

[0492] Bacterial Transfection and Checking of the Construct

[0493] The plasmid thus obtained is transfected into E. coli D115 a.Furthermore, the DNA of the plasmid is extracted and the heterologousinsert is sequenced.

[0494] Cell Culture, Extraction and Purification of the RecombinantProtein

[0495] The recombinant bacterial cells are cultured at 37° C. in an LBmedium containing antibiotics until the OD at 600 nm reaches 0.2. Theproduction of recombinant protein is then induced by adding 1 mMisopropyl-p-D-thiogalactopyranoside to the culture medium. The bacterialculture is continued for 16 h at 37° C. The cells are recovered bycentrifugation. The cells are lysed using lysozyme in a Tris buffer pH7.4 in the presence of NaCl, PMSF and sodium deoxycholate. The DNA isdegraded by sonication. After centrifugation, the recombinant protein isseparated from the supernatant using a Probond column (Invitrogen,France). This column contains charged nickel which has affinity for thehexahistidine peptides. The elution is carried out in the presence ofimidazole. The protein concentration is estimated by the Lowry methodafter having dialysed the product of the elution. The purity of theprotein obtained is tested by SDSPAGE electrophoresis, which shows asingle band.

Example 1 Identification of the Protein Complex Responsible for the LSRActivity: Partial Purification and Characterization By Means ofPolyclonal Antibodies

[0496] The technique of ligand blotting was used to identify theproteincomplex responsible for the LSR activity. This technique,described in detail by Mann et al., 1995, is detailed below.

[0497] Ligand Blotting

[0498] The technique consists in isolating, by differentialcentrifugation (Belcher et al., 1987) rat liver membranes, and insolubilizing the membrane proteins in a solution containing 125 mMoctylglucoside, 20 mM Tris and 2 mM EDTA, pH 8. The proteins thussolubilized are separated under nondenaturing conditions on apreparative SDS gel (thickness 5 mm) consisting of a gradient from 4 to12% polyacrylamide (35-50 mg of protein per gel). For part of the gel,the proteins are then electrotransferred (semi-dry transfer, 21 V, 45min, Biorad) onto a nitrocellulose membrane. After blocking the freesites of this membrane in a PBS solution containing 3% albumin, themembrane is incubated with 40 μg/ml of ¹²⁵I-LDL in the presence (FIG. 8,lane 2) or in the absence (FIG. 8, lane 1) of 0.8 mM oleate. Themembrane is then washed five times for 10 minutes in PBS containing 0.5%(v/v) Triton X100, and exposed on a Phosphor Imager screen.

[0499] Analysis of the image obtained in the presence (FIG. 8, lane 2)or in the absence (FIG. 8, lane 1) oleate shows the presence of 3 mainbands which have bound the LDLs. The apparent MW of the first band isabout 240 kDa, that of the second is 115 kDa and that of the third is 90kDa. On the basis of these observations, two hypotheses are formulated:on the one hand, the LSR activity is linked to the presence of severaldistinct proteins; on the other hand, the same type of image can beexplained by a multimeric organization of a protein complex.

[0500] In order to check this hypothesis, the inventors undertook thepurification of the band having the highest apparent molecular weight(240 kDa). The partial purification of this protein, designated “bandA”, is carried out by preparative electrophoresis as follows.

[0501] Partial Purification of LSR

[0502] The technique consists in isolating, by differentialcentrifugation (Belcher et al., 1987) rat liver membranes, and insolubilizing the membrane proteins in a solution containing 125 mMoctylglucoside, 20 mM Tris and 2 mM EDTA, pH 8. The proteins thussolubilized are separated under nondenaturing conditions on apreparative SDS gel (thickness 5 mm) consisting of a gradient from 4 to12% polyacrylamide (35-50 mg per gel). For part of the gel, the proteinsare then electrotransferred (semi-dry transfer, 21 V, 45 min, Biorad)onto a nitrocellulose membrane. After blocking the free sites of thismembrane in a PBS solution containing 3% albumin, the membrane isincubated with 40 μg/ml of ¹²⁵I-LDL in the presence (FIG. 8, lane 2) orin the absence (FIG. 8, lane 1) of 0.8 mM oleate. The membrane is thenwashed five times for 10 minutes in PBS containing 0.5% (v/v) TritonX100, and exposed on a Phosphor Imager screen. The proteins of interestare electroeluted (Eletroeluter, Biorad).

[0503] The rat liver plasma membrane proteins were prepared andseparated on a polyacrylamide gel as above. The precise location of bandA was established by ligand blotting carried out after electrotransferof preprative gel sample removed at various levels.

[0504] The gel fragments containing band A are then collected,electroeluted and concentrated (speedvac), and then tested for theircapacity to bind the LDLs in the presence of oleate afterelectrophoresis and transfer onto nitrocellulose membranes (FIG. 8, lane3; 80 μg of protein/lane).

[0505] The proteins thus obtained were also used to produce polyclonalantibodies whose specificity was tested by Western blotting (FIG. 8,lane 4).

[0506] Preparation of Polyclonal Antibodies

[0507] The LSR proteins used as antigens for the production of anti-LSRantibodies were prepared as indicated above.

[0508] The antigen preparation is injected subcutaneously into a rabbitin the presence of complete Freund's adjuvant, followed by aconventional immunization procedure. The titer of the antibody directedagainst the rat proteins is determined regularly (dot-blot technique).When the latter is judged to be sufficient, the specificity of theantibodies obtained is tested by Western blotting on a preparation ofsolubilized proteins of rat liver membranes as described above, withanti-rabbit IgG goat antibodies labelled with iodine I¹²⁵ as secondantibodies.

[0509] The Western blot results after electrophoresis under nonreducingconditions indicate that the antibodies produced from the proteins ofband A bind to 3 main protein bands (240 kDa, 115 kDa and 90 kDa) whichbind the ¹²⁵I-LDL in the presence of oleate (FIG. 8, lane 4). To verifythe link between these protein complexes and the LSR activity, theeffect of these polyclonal antibodies on the LSR activity was tested.

[0510] The methods used are described in detail below (Mann et al.,1995; Troussard et al., 1995). The LSR activity is estimated bymeasuring the binding of lipoproteins to plasma membranes and bymeasuring the binding, internalization and degradation of thelipoproteins on primary cultures of rat hepatocytes.

[0511] Measurement of the Binding of Lipoproteins on Plasma Membranes

[0512] The LSR activity is measured on a preparation of rat liver plasmamembranes (Bartles and Hubbard, 1990). These membranes exhibit 10 to15-fold enrichment with 5-nucleotidase (marker specific for plasmamembranes). 100 μg aliquots of proteins are incubated for 30 minutes at37° C. in the presence or in the absence of 0.8 mM oleate in a finalvolume of 250 μl supplemented with 100 mM PBS, 2 mM EDTA, 350 mM NaCl,pH 8 (buffer A). The oleate is added in a volume of 5 to 10 μl ofisopropanol. The excess and unbound oleate is then removed by 6 washes.The pellets are resuspended in 250 μl of incubation buffer, sonicatedfor 5 seconds, power 1.90% in the active cycle, and then centrifuged for15 min at 18,000 rpm. The activated membranes are incubated for 1 hourat 4° C. with various concentrations of antibody and then with 5 μg/mlof ¹²⁵I-LDL (1 hour at 4° C.). 25 μl of 2% BSA are added to theincubation mixture. The quantity of ¹²⁵I-LDL bound to the membranes ismeasured by sedimenting the membranes by centrifugation after havingdeposited 200 μl of the incubation mixture on a layer of 5% (WN) of BSAin buffer A. The supernatants are removed by aspiration, the tubebottoms are cut off and their radioactivity is counted in a y counter.

[0513] The inhibitory effect of anti-LSR antibodies on the LSR activity,compared to that of any preparation of rabbit immunoglobulins is shownin FIG. 9A. The inhibition of the LSR activity by the anti-LSRantibodies confirms that the multimeric complex described above isresponsible for the activity of the receptor and validates the ligandblotting technique used to identify it. The effect of the anti-band Aantibodies was, in addition, tested on the other steps of the activityof the receptor: the internalization and the degradation of lipoproteinsby the LSR expressed at the surface of hepatocytes in primary cultures.

[0514] Measurement of the Binding, Internalization and Degradation ofLipoproteins by Hepatocytes

[0515] The LSR activity in the primary cultures of rat hepatocytes ismeasured by the binding, internalization and degradation of I¹²⁵I-LDLand ¹²⁵I-VLDL (LDL: low-density lipoprotein; VLDL: very low-densitylipoprotein), as described in Bihain and Yen, 1992 and Mann et al.,1995.

[0516] To measure the effect of the anti-LSR antibodies on the binding,internalization and degradation of LDLs by LSR, primary cultures of rathepatocytes (48 h after plating) are incubated in the presence of 20 ngof leptin/well for 30 min at 37° C., followed by the addition ofanti-LSR antibodies in the presence or in the absence of oleate. Afterincubating at room temperature for 30 min, ¹²⁵I-LDL (20 μg/ml) is addedand then the cells are incubated for 4 h at 37° C. The binding,incorporation and degradation of ¹²⁵I-LDL are measured as described inBihain and Yen, 1992 and Mann, et al., 1995.

[0517] The data in FIG. 9B show that the anti-band A antibodies inhibitmost of the activity of binding of the LDLs to the LSRs present at thelevel of the hepatocytes. This inhibition induces a decrease in the sameproportions in the internalization and proteolytic degradataion of thelipoproteins.

[0518] The anti-band A antibodies are thus characterized as anti-LSR.Their relative specificity was defined by a selectiveimmunoprecipitation method. Extracts of hepatocytes in primary cultureare immunoprecipitated by means of the anti-LSR antibodies describedabove, according to the protocol described below.

[0519] Immunoprecipitation of Extracts of Hepatocytes in the Presence ofSpecific Antibodies

[0520] Primary cultures of rat hepatocyte (Oukka et al., 1997) areincubated for 60 minutes to 2 hours in the presence of a mixture of³⁵S-methionine and ³⁵S-cysteine (Promix, Amersham). This medium is thenremoved and the cells are washed and then incubated in PBS containing 1%of Triton X100. This cellular lysate is then incubated in the presenceof non-specific antibodies and then of protein A. The equivalent of 40μg of specific anti-LSR antibodies is then added and the LSR-antibodycomplexes are precipitated with the aid of a second preparation ofprotein A. After washing, the complexes are dissociated in the presenceof 1% SDS supplemented or otherwise with 5% P-mercaptoethanol, incubatedat 100° C. for 5-10 minutes, and separated on a 10% acrylamide gel. Thegels are dried and exposed on a Phosphor Imager screen. Each of thelanes contains the equivalent of a 165 cm² flask, that is to say 22×10⁶cells.

[0521] Analysis of the immunoprecipitation results indicates that undernonreducing conditions (FIG. 10, lanes 2—without incubation at 100° C.-and 3—with incubation at 100° C.-), the antibodies reveal 3 principalprotein bands: 2 of apparent molecular weight 240 kDa and 180 kDa, 1 ofapparent molecular weight 68 kDa. The presence of 2 bands of weakerintensity, corresponding to a molecular weight of 115 kDa and 90 kDa,can also be noted. This experimental approach therefore essentiallyidentifies the same protein elements as those identified by the ligandblotting method. It can be observed, moreover, that under reducingconditions (FIG. 10, lanes 1 and 4), the elements of high molecularweight dissociate into 3 elements of apparent molecular weight 68 kDa,56 kDa and 35 kDa, respectively.

[0522] The relative intensity of the 68 kDa and 56 kDa bands is similarwhereas that of the 35 kDa band is about ¼ of that of the other two.

Example 2 Cloning of the c-DNA Encoding the α- and β-LSR

[0523] The screening of an expression library by means of the anti-LSRantibodies described above was carried out as indicated below.

[0524] Screening of an expression library

[0525] After infection of bacteria with lambda GT11 bacteriophagescontaining rat liver cDNA (commercially obtained from ClontechLaboratories Inc.) (5′ Strech Plus c-DNA Library), the cells are platedon LB MgSO₄ medium. After 4 hours of culture at 42° C., a nitrocellulosemembrane, previously incubated in a 10 mM IPTG solution, is deposited inthe Petri dishes. Four hours later, the first membrane is removed and asecond is applied to the Petri dish.

[0526] Each membrane is immersed in a Petri dish containing blockingbuffer kept stirring for one hour. Next, the antibody is added to afinal concentration of 10 g/ml of blocking buffer (Huynh et al., 1984;Young and Davis, 1983a and 1983b). The membranes are then washed threetimes for 10 minutes with TNT (10 mM Tris, 150 mM NaCl, 0.05% Tween 20).

[0527] The membranes are incubated in the presence of secondaryantibodies (alkaline phosphatase-conjugated affinipure F(ab′) 2 fragmentgoat anti-rabbit IgG; Immunotech) at a final concentration of 0.08 μg/mlof blocking buffer (TNT+5% powdered skimmed milk, Pâturage trademark).

[0528] After washing the membranes in TNT, they are incubated in thepresence of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and of NBT(nitro blue tetrazolium) until a colour is obtained.

[0529] The positive clones are then recovered on the dishes, titratedand subjected to the same immunoscreening procedure so as to confirmthat they are true positives (secondary screening). Optionally, atertiary screening may be carried out. The phage DNA of the selectedclones, isolated from a bacterial lysate (Clontech protocol), anddigested with the restriction enzyme EcoR1 is inserted at the EcoR1 siteof the plasmid pBluescript SK+.

[0530] Two clones containing an insert of 1.8 kb were thus obtained, andproved to be of identical sequences. The hybridization of rat liver mRNA(2 μg of polyA+ mRNA) with a probe corresponding to the BgIII-XbaIfragment of this insert revealed two bands of sizes 1.9 kb and 2.1 kb(FIG. 11A) respectively. Northern blot analysis, with a probecorresponding to the XbaI-XbaI fragment of this insert, of the tissuedistribution of the corresponding messengers showed that they arepreferably expressed in the liver (FIG. 11B). The Nothern blotting wascarried out according to the following protocol.

[0531] Northern Blotting

[0532] The membranes containing the mRNAs of different rat tissues(Clontech) were hybridized with fragments of the cDNA for the LSR geneand of the cDNA for human β-actin (Clontech), labelled with [³³P]dCTP,in 5×SSPE, 10× Denhardt buffer containing 0.5% SDS, 100 μg/ml of salmonsperm DNA, 50% deionized formamide, at 42° C. for 16 hours. Themembranes were then washed in 2×SSC, 0.5% SDS at room temperature and in1×SSC, 0.1% SDS at 65° C., and then exposed on the Phospor Imager(Molecular Dynamics).

[0533] A cDNA corresponding to the 1.9 kb band was reconstructed by5′RACE PCR from the 1.8 kb fragment and sequenced.

[0534] In order to elucidate the presence of multiple bands in Northernblotting, several pairs of primers defining fragments of a rat cDNAsequence were synthesized and used as primers for a PCR amplification(FIG. 11C). The sequences of the oligonucleotides used are listed below:a: 5′-GTTACAGAATTCGCCGCGATGGCGCCGGCG-3′ (SEQ ID 20) b:5′-GCCAGGACAGTGTACGCACT-3′ (SEQ ID 21) c: 5′-ACCTCAGGTGTCCCGAGCAT-3′(SEQ ID 22) d: 5′-GAAGATGACTGGCGATCGAG-3′ (SEQ ID 23) e:5′-ACCTCTATGACCCGGACGAT-3′ (SEQ ID 24) b′: 5′-CACCACCCTGACAGTGCGTA-3′(SEQ ID 25) c′: 5′-CTGGGGGCATAGATGCTCGG-3′ (SEQ ID 26) d′:5′-GCCCTGGAAGGCCTCGATCG-3′ (SEQ ID 27) e′: 5′-CAAGTCCCTAGGATCGTCCG-3′(SEQ ID 28)

[0535] Whereas each pair of primers shows a single fragment, the bc′pair makes it possible to amplify three fragments of different sizes.Analysis of the sequences of these fragments makes it possible toreconstitute the sequence of three complete cDNAs for rat LSR, havingsizes of 2097 bp (SEQ ID 1), 2040 bp (SEQ ID 3) and 1893 bp (SEQ ID 5)respectively, and all three corresponding to the same precursormessenger by alternative splicing.

[0536] These three cDNAs contain an open reading frame starting with anAUG codon at position 219 surrounded by a Kozak consensus sequence(Kozak, 1987 and 1990). The predicted molecular weights of the proteinsencoded by these three cDNAs are 66 kDa, 64 kDa and 58 kDa,respectively.

[0537] The two cDNAs encoding respectively the longest and the shortestforms of rat LSR were then translated in vitro as indicated below.

[0538] Translation In Vitro

[0539] The cDNAs are subcloned into the plasmid pcDNA3; transcriptionand translation in vitro are carried out using the Promega TNT kit. Theproducts of translation, labelled with ³⁵S-methionine and ³⁵S-cysteine,are visualized after electrophoresis on a polyacrylamide gradient gel(10%) and exposure on Phospor Imager.

[0540] The molecular weights of the products obtained, that is to say 68kDa and 56 kDa (FIG. 12), correspond closely to those of the a and βsubunits of LSR.

[0541] To define if the products of these mRNAs are responsible for thereceptor activity, three different experimental approaches were used.

[0542] Firstly, two peptides corresponding to residues 169-186(SAQDLDGNNEAYAELIVLGR: SEQ ID 29) of the LSR produced from the mRNA ofsize 2097 bp and to residues 556-570 (EEGQYPPAPPPYSET: SEQ ID 30) weresynthesized. The sequence of these peptides is common to the threeproteins identifed above. Antibodies directed against these syntheticpeptides were obtained according to the protocols indicated above. FIGS.13C and 14C show that these anti-LSR peptide antibodies have aninhibitory effect on the binding of the LDLs to the LSRs present on ratplasma membranes, measured according to the protocol described inExample 1.

[0543] Secondly, a partial purification of the α and β subunits wasobtained by selective solublization with the aid of sarkosyl; a studyusing Western and ligand blotting showed that the α and β componentsbind the anti-LSR polyclonal antibodies (FIG. 13B, lane 1), the anti-LSRpeptide antibodies (FIG. 13B, lane 2 and FIG. 14B, lane 2), and the LDLsafter incubation with oleates (FIG. 13B, lane 4). Ligand blotting wascarried out according to the protocol described in Example 1; Westernblotting was carried out as indicated below.

[0544] Western Blotting

[0545] Primary cultures of rat hepatocytes are prepared as indicated in“Experimental procedures ” The cells harvested after 48 hours of cultureare washed and lysed in PBS containing 1% Triton X100. The lysates aredeposited on a 10% SDS-PAGE gel under reducing conditions (2% SDS, 5%β-mercaptoethanol and 20 mM DTT, at 56° C. for 1 h). After transferringonto a nitrocellulose membrane, the Western blotting is carried out withIgG antibodies directed against the LSR receptor.

[0546] Thirdly, the labelled proteins LSR 66 and 58 obtained by in vitrotranslation from the cDNAs LSR-Rn-2097 and LSR-Rn-1893 are used toestimate the effect of oleate on the binding of the LDLs according tothe protocol detailed below.

[0547] Binding of the LDLs onto the LSR Proteins Expressed In Vitro(“Flotation ”)

[0548] The ³⁵S-cysteine or ³⁵S-methionine labelled products oftranslation in vitro (17 μl) are incubated for 1 hour at 37° C. in thepresence of 100 μg/ml of LDL, 1 mM oleate in buffer A, in a final volumeof 400 μl. An equal volume of 8% (w/v) BSA is added. The density isadjusted to 1.21 g/ml (assuming an initial density of 1.025 g/ml), withsodium bromide. The samples are then deposited on a sodium bromidesolution at 1.063 g/ml, and then centrifuged for 20 hours at 4° C.(Beckman SW41 rotor). A volume of 1 ml is collected at the surface,dialysed against electrophoresis elution buffer, and the radioactivityis counted (Beckman β counter).

[0549] Oleate increases the binding of LDL to LSR 56 (respectively LSR68) by a factor of 2 (5 respectively). It can thus be shown that the aand β subunits of rat LSR, encoded respectively by the cDNAs LSR-Rn-2097and LSR-Rn-1893 (LSR 56 and LSR 68), preferably bind the LDLs afterincubation with oleate.

[0550] All these results indicate that the cDNAs LSR-Rn-2097 andLSR-Rn-2040 encode two proteins which are indistinguishable byelectrophoresis and whose apparent molecular weight is 68 kDa; theseproteins correspond to the band comprising the a and α′ subunits of LSR,which is identified after immunoprecipitation under reducing conditions.The β subunit of LSR is presumably the product of translation of thecDNA LSR-Rn-1893. The analyses of stoichiometry afterimmunoprecipitation indicate that the multimeric complex of apparentmolecular weight 240 kDa is the result of an assembly of an α subunitwith three β subunits. Analysis of the various domains of the proteinscorresponding to the α and β-LCRs is compatible with a lipoproteinreceptor function.

Example 3 Analysis of the Activity of a Recombinant LSR Receptor, andits Subunits, in Transfected Cells

[0551] The inventors also expressed a recombinant LSR receptor in CHOcells according the following protocol.

[0552] Transfection with cDNA Sequences Encoding the LSR Receptor

[0553] In order to study the activity of each of the recombinantsubunits of LSR, as well as the activity of a reconstituted receptor,the inventors used the expression plasmid pcDNA3 (No et al., 1996) tostudy the expression, in animal cells, of either cDNA encoding the αsubunit (β plasmid), or of a cDNA encoding the β subunit (β plasmid), ofrat LSR. The LSR cDNAs were subcloned into the plasmid pcDNA3(Invitrogen) using the EcoRI and/or NotI restriction sites. Onceobtained, these constructs are used to transfect CHO (Chinese hamsterovary) animal cells.

[0554] After 48 hours of culture, CHO (Chinese hamster ovary) cells(CHO-K1, CCL-61, ATCC, Rockville, Md.) were distributed into 6-wellplates (Falcon) at 2.5-2.75×10⁵ cells/well. After 24 h of culture in aHam F-12 medium containing 10% (v/v) FBS, 2 mM glutamine and 100units/ml of penicillin and streptomycin, a maximum of 2 μg ofplasmid/well were transfected using Superfect (Qiagen) according to thesupplier's instructions (10 μl Superfect/well, 2 h at 37° C. in a HamF-12 medium free of serum). The plates were then washed in PBS in orderto remove the transfection reagents and the cells were then cultured ina Ham F-12 medium containing serum. The LSR activity was measured 48 hafter transfection according to the protocols detailed in Example 1.

[0555] The inventors tested the effect of a co-transfection with the aand β plasmids compared with that of a transfection with the α plasmidalone, or with the β plasmid alone, on the three stages of the activityof the LSR receptor according to the protocols detailed below. FIGS. 15and 16 show the comparisons between the LSR activities obtained on therecombinant cells expressing the α subunit alone, or the two α and βsubunits; similar results are obtained for the β versus α+β comparison,which is compatible with the comparative analysis of the primarysequences of each of the subunits (each of them also carrying thepotential binding sites for lipoprotein ligands and fatty acids, such asoleate).

[0556] Effect of a Transfection With the LSR (a) Plasmid Alone, or of aCo-Transfection With the LSR (α) and LSR (β) plasmid, on the binding,internalization and degradation of the LDLs

[0557] The CHO-K1 cells were transiently transfected with increasingconcentrations of α plasmid and co-transfected with 0.4 μg of α plasmidand increasing concentrations of β plasmid. After 48 h of culture, thecells were washed once with PBS and incubated for 3 h at 37° C. with 20μg/ml ¹²⁵I-LDL in the presence or in the absence of 1 mM oleate in DMEMcontaining 0.2% BSA, 5 mM Hepes, and 2 mM CaCl₂, pH 7.5. Next, the cellswere washed as described above and incubated at 4° C. for 1 h with 10 mMsuramin in PBS.

[0558] To measure the binding of the LDLs (FIG. 15), the medium wasrecovered and passed through a γ counter in order to evaluate thequantity of bound ¹²⁵I-LDL. The results are the mean values of twomeasurements. For the measurement of the internalization and thedegradation of LDLs (FIG. 16), the quantity of ¹²⁵I-LDL internalized anddegraded was measured according to the protocols detailed in Example 1.

[0559] The co-transfection with a and β plasmids makes it possible toestablish three stages of LSR activity (FIGS. 15 and 16).

[0560] The inventors also observed that the co-transfection with the αand β plasmids increases the LSR activity compared with a transfectionwith only an α plasmid. The results suggesting a more efficient activityof the LSR when the ([P]/[a]) ratio between the concentrations of β andα subunits expressed increases, is compatible with the observation thatthe LSR receptor might consist of the assembly of an α (or α′) subunit,and of several, probably three, β subunits.

[0561] The results show that only the co-transfection of the β and asubunits allows the overexpression of a completely functional LSRreceptor in the sense that it allows the complete proteolyticdegradation of the protein.

[0562] In order to characterize the lipoprotein degradation activityobtained above in cells transfected with the LSR cDNAs, the inventorsfinally tested the capacity of anti-LSR antibodies to inhibit thebinding of LDLs as measured above, as well as the substrate-specificitythereof.

[0563] Characterization of the Lipoprotein Degradation Activity Obtainedin Transfected Cells Expressing a Recombinant LSR Receptor

[0564] The CHO cells were transfected with the a end β plasmids in aconcentration ratio of 1 to 3.

[0565]FIG. 17A shows that the LDL binding activity obtained in thetransfected cells (expressed relative to the same activity observed innontransfected control cells) is specifically inhibited by the anti-LSRantibodies.

[0566]FIG. 17B shows the LDL binding activity obtained in the cellstransfected in the presence of various nonlabelled lipoproteins actingas competitive ligands. The results show a ligand specificity similar tothat observed for the endogenous LSR activity in rats (Mann et al.,1995): the rat chylomicrons are the preferred substrates for the ratrecombinant LSR; then come in particular, in decreasing order ofspecificity, the VLDLs and then the LDLs.

Example 4 Involvement of LSR in the Clearance of Cytokines

[0567] The analysis of the sequence of the α subunit of LSR reveals acysteine-rich region which corresponds to a Tumor Necrosis Factor typecytokine receptor signature. LSR is, however, distinguishable from thecytokine receptors by the presence of signals allowing rapid endocytosisof the receptor/ligand complex (clathrin motif).

[0568] The inventors formulated the hypothesis that this receptor couldserve for the removal of cytokines, and in particular of leptin; inorder to verify this hypothesis they analysed the degradation ofrecombinant leptin by hepatocytes in primary culture according to theprotocol below.

[0569] Degradation of Leptin by Hepatocytes in Primary Culture

[0570] Primary cells of rat hepatocytes are incubated for 4 hours at 37°C. with 20 ng/ml of ¹²⁵I-leptin in the absence or in the presence of 0.5mM oleate, 75 μg/ml of RAP, 200 μg/ml of non-specific antibodies oranti-LSR specific antibodies, or 50 μM chloroquinine. The medium is thenrecovered and the quantity of ¹²⁵I-leptin degraded is measured.

[0571] As indicated in FIG. 18, the degradation of leptin by hepatocytesin primary culture is inhibited by:

[0572] a) polyclonal antibodies directed against LSR. These antibodiesalso inhibit, in the same proportions, the LSR activity,

[0573] b) the 39 kD Receptor Associated Protein (RAP); this proteinblocks the LSR activity in vitro and retards the clearance ofchylomicrons in vivo (Troussard et al., 1995; Willow et al., 1994)

[0574] c) chloroquine; this cellular poison prevents the acidificationof the endocytosis vesicles and inhibits the activity of the lysosomalproteases,

[0575] d) oleate; this free fatty acid induces a change in theconformation of LSR which unmasks the lipoprotein binding site.

[0576] This indicates that the FAF (Fatty Acid Free) conformation of LSRis probably the only one which is compatible with the role of bindingfollowed by degradation of leptin. The non-specific immunoglobulins arewithout effect on the degradation of leptin (FIG. 18).

[0577] In order to verify the binding of leptin to LSR, the rat liverplasma membrane proteins were deposited on an affinity chromatographycolumn containing recombinant leptin, according to the protocol detailedbelow.

[0578] Leptin Affinity Chromatography

[0579] A Hi-trap column (Pharmacia) is used: 5 mg of leptin are boundonto 1 ml of column according to the methods recommended by themanufacturer. The plasma membrane proteins are solubilized from ratlivers as indicated above (Mann et al., 1995), and then dialysedovernight against PBS pH 7.4, 0.1% Tween 20. The column is washed in thesame buffer and the protein extract is deposited at a rate of 0.2ml/minute. The column is washed with 6 ml of the same buffer. It is theneluted with the same buffer supplemented with 100 mM glycine pH 3; 20fractions of 500 μl are then neutralized with 5 μl of PBS, 0.1% Tween20, pH 8. 50 μl of each fraction are deposited on a nitrocellulosemembrane for dot-blot analysis by means of anti-LSR antibodies. Thepositive fractions (1, 3, 4, 7 and 8) are dialysed against 24 mMammonium bicarbonate, 0.01% Tween 20, pooled and concentrated in aSpeedvac in a final volume of 300 μl. 40 μl of the final product areanalysed by Western blotting by means of anti-LSR antibodies.

[0580]FIG. 19 shows that the anti-LSR antibodies specifically recognizethe α subunit which, after binding to leptin, was released by theglycine buffer.

[0581] Experiments of stable transfection of the α subunit will make itpossible to measure the affinity of leptin for this new receptor.

[0582] All these results suggest that LSR represents one of the pathwaysfor the degradation and elimination of leptin. The in vivo injection ofradiolabelled recombinant leptin showed, both in the obese mice and inthe control mice, a rapid speed of clearance and a preferential captureof leptin by the liver and the kidney: 50% of the injected dose is foundafter 10 minutes in these two organs. In order to analyse the mechanismsfor the selective capture of leptin, the inventors compared thequantities of leptin and of 2 microglobulin (soluble protein having amolecular weight close to that of leptin, chosen as control) present inthe kidney and liver of normal mice and of two obese mouse lines 5minutes after injection of the same tracer dose of these tworadiolabelled proteins.

[0583] Measurement of the Clearance of Leptin in Mice

[0584] The female control, ob/ob, or db/db mice (6-8 weeks), on an emptystomach, are anaesthetized and receive via the saphenous vein aninjection of 80 ng of murine recombinant ¹²⁵I-leptin or of¹²⁵I-β₂-microglobulin (Sigma, labelled by the Iodobeads method, likeleptin). Five minutes later, the animals are infused with aphysiological saline solution (15 ml, at 4° C.). The tissues arecollected and counted for their radioactivity (Gamma counter). In somecases, an anti-LSR antibody or a control protein are injected 30 minutesafter injection of ¹²⁵I-leptin. It is important to note that thelabelling of leptin with ¹²⁵I has no effect on its biological activity.

[0585] The results presented in FIG. 20 show that the quantity of leptinselectively captured by the liver is reduced in the obese mice, comparedwith the control mice; moreover, no difference is observed between thevarious lines as regards the renal capture of leptin.

[0586] The inventors then measured the number of LSR receptors incontrol, ob/ob and db/db mice according to the following protocol.

[0587] Measurement of the Apparent Number of LSR Receptors on PlasmaMembranes

[0588] The apparent number of LSR receptors on plasma membranes ismeasured as previously described (Mann et al., 1995) by estimating thequantity of LDL bound to a plasma membrane preparation. The plasmamembranes (100 μg) are incubated with 1 mM oleate; they are then washedthree times as indicated above, and then incubated for 1 hour at 37° C.with 40 μg/ml of ¹²⁵I-LDL. The quantity of ¹²⁵I-LDL bound to the plasmamembranes is then determined by counting. The mean is established on 3measurements per animal for 3 different animals in each of the groups.

[0589]FIG. 21 shows that the number of LSR receptors in obese animalsexhibiting either a deficiency in leptin (ob/ob), or a deficiency in theob receptor (db/db), is significantly reduced. The reduction in theselective hepatic capture of leptin in obese mice coincides with thereduction, in these animals, of the apparent number of LSR receptors.

[0590] The inventors finally tested, according to the protocol presentedbelow, the effect of anti-LSR antibodies on the distribution of leptinbetween the liver and the kidney, 5 minutes after injection of a tracerdose.

[0591] Measurement of the Distribution of Leptin Between the Liver andthe Kidney in the Presence of anti-LSR Antibodies

[0592] Control mice are anaesthetized and then they are injectedintravenously with 1 mg of non-specific IgG antibody or of anti-LSR IgGantibody. After 30 minutes, 80 ng of ¹²⁵I-leptin are injected and, after5 minutes, an infusion of physiological saline solution at 4° C. Thetissues are removed immediately and the radioactivity is measured. Theresults represent the mean and the standard deviation obtained for 3animals for each of the groups.

[0593] As shown in FIG. 22, the hepatic capture of leptin is reduced andthe renal capture is increased by the anti-LSR antibodies, compared withthe control immunoglobulins.

[0594] These results therefore indicate that LSR is responsible for theselective hepatic capture of leptin and that a reduction in the numberof receptors is observed in the obese animals. Such a reduction mayexplain the leptin-resistance syndrome and the increase in the plasmaconcentration of leptin which is observed in most obese human subjects.

[0595] It is also possible that the LSR receptor serves as degradationpathway for other cytokines, in particular those produced by the adiposetissue. The importance of Tumor Necrosis Factor a and Nerve GrowthFactor will be noted in particular. These two cytokines exert asignificant slimming effect when they are injected into human subjects(Cytokines and their receptors, 1996).

Example 5 Control of the LSR activity by cytokines

[0596] The α subunit of the LSR receptor binds leptin and possessespotential phosphorylation sites. This makes it a receptor which not onlymediates endocytosis, but could also serve in cell signalling.

[0597] The inventors therefore tested the hypothesis according to whichleptin modulates the activity of LSR, as described below.

[0598] Measurement of the LSR Activity of Binding, Internalization andDegradation of Lipoproteins in the Presence of Leptin

[0599] Rat hepatocytes in primary culture are incubated at 37° C. for 30min with an increasing concentration of leptin, and then incubated at37° C. for 4 hours with either 50 μg/ml of ¹²⁵I-LDL (specific activity:209 cpm/ng) or 50 μg/ml of ¹²⁵I-VLDL (specific activity: 157 cpm/ng) inthe absence or in the presence of 500 μM oleate. The cells are thenwashed and the quantities of 1-lipoproteins bound, incorporated anddegraded are measured as described above in Example 1 (Bihain and Yen,1992). The results shown in FIG. 23 represent the differences obtainedbetween the cells incubated with or without oleate. Each pointrepresents the mean of 3 measurements. The standard deviation for eachpoint is included in the symbol.

[0600] The addition of increasing concentrations of leptin tohepatocytes in culture increases the binding, internalization anddegradation of VLDLs and LDLs (FIG. 23).

[0601] Analysis of the Capacity for Inducing the LSR Activity by Leptin

[0602] Measurement, in the Presence of Leptin, of the Apparent Number ofLSR Receptors Expressed at the Surface of Rat Hepatocytes in PrimaryCulture

[0603] Primary cultures of rat hepatocytes are incubated for 30 min at37° C. in the presence or in the absence of 20 ng/ml of leptin, for 10min at 37° C. in the presence of 0.8 mM oleate. The cells are washedwith PBS buffer precooled to 4° C., and then incubated for 2 hours at 4°C. in the presence of increasing concentrations of ¹²⁵I-LDL. The cellsare then washed, lysed and the quantity of bound ¹²⁵I-LDL is measured.

[0604] Comparative Effects of Leptin in the Presence of Cycloheximide,Colchicine and Cytochalasin B

[0605] The initial conditions are identical to those described above;after incubation with leptin, the cells are incubated for 30 min at 37°C. with 5 μM cycloheximide, 5 μM colchicine or 2.5 μM cytochalasin B.The cells are then incubated for 10 min at 37° C. in the presence of 0.8mM oleate. The cells are then washed with PBS buffer precooled to 4° C.,and then incubated for 2 hours at 4° C. in the presence of 50 μg/ml of¹²⁵I-LDL. 2 measurements are carried out, and the mean results arepresented.

[0606] It is thus shown that the increase in the LSR activity by leptinis obtained through an increase in the apparent number of receptorsexpressed at the surface of the hepatocytes (FIG. 24A). This increaseresults, on the one hand, from an increase in protein synthesis (it ispartially inhibited by cycloheximide, an inhibitor of proteinsynthesis). It involves, on the other hand, the mobilization of theendocytosis vesicles by the microtubule system (it is indeed inhibitedby cytochalasin B which blocks microtubular transport) (FIG. 24B).

[0607] In order to check the in vivo effect of leptin on the LSRactivity, the inventors characterized the postprandial triglyceridernicresponse of control, ob/ob and db/db mice after a force-fed test mealaccording to the following protocols. Measurement of the postprandiallipemic response in mice Control, ob/ob and db/db mice, starved sincethe day before, are force-fed with a meal which is very high in fat [60%fat (37% saturated, 27% monounsaturated and 36% polyunsaturated fattyacids), 20% protein and 20% carbohydrate] providing 56 kcal of energy/kgof the weight of the animal. Immediately after the meal (time=0 hour),the mice are injected intravenously with 200 μl of physiological salinesolution. At various times, 20 μl of blood are collected via the caudalvein in tubes containing 90 μg of disodium EDTA, and after separatingthe plasma by centrifugation, the plasma concentration oftriglyceridernia is determined with the aid of an enzymatic assay kit.Each point on the curves presented corresponds to the mean with standarddeviation obtained for 3 measurements per animal and for 3 differentanimals.

[0608] Measurement of the Effect of Leptin on the Postprandial LipemicResponse in Mice

[0609] The procedure is the same as above, except that immediately afterthe meal (time=0 hour), the mice are injected intravenously with either200 μl of physiological saline solution, or 200 μl of the same solutioncontaining 50 μg of murine recombinant leptin. Measurement of thepostprandial lipemic response in mice in the presence of lactoferrinand/or leptin ob/ob mice, starved since the day before, are force-fedwith a meal identical to that described above. Immediately after themeal (time=0 hour), the mice are injected intravenously with 200 μl ofsaline solution containing either no supplement, or 0.5 μg of leptin, or2.5 mg of lactoferrin or alternatively a mixture of 0.5 μg of leptin and2.5 mg of lactoferrin. Blood is collected between 2 and 3 hours afterthe meal and the plasma concentration of triglycerides (TG) is measured.The values obtained represent the mean with standard deviation obtainedfor 4 measurements per animal and for 2 different animals [p<0.02 (ob/obcompared with ob/ob+leptin), p<0.01 (ob/ob compared withob/ob+lactoferrin), NS (ob/ob+lactoferrin compared withob/ob+leptin+lactoferrin)].

[0610] In agreement with the reduction in the number of LSR receptorsobserved in the obese mice, an amplification of the postprandial lipemicresponse also exists in the untreated obese mice. The administration ofleptin by the intravenous route, at the same time as the test meal,makes it possible to reduce the postprandial lipemic response in the twoobese mouse lines and in the control mice (FIG. 25).

[0611] This reduction in the lipemic response induced by leptin issuppressed by the administration of lactoferrin (FIG. 26), which blocksthe activity of LSR (Yen et al., 1994; Mann et al., 1995). This stronglysuggests that the reduction in the lipemic response is explained by anincrease in the LSR activity.

[0612] Finally, also in vivo, the administration of leptin induces anincrease in the apparent number of LSR receptors expressed at the levelof the surface of the hepatocytes. This increase is significant both inthe ob/ob mice and in the db/db mice (FIG. 27).

[0613] Leptin and probably other cytokines are therefore regulators ofthe activity of LSR. A syndrome of resistance to leptin or to othercytokines can lead to hypertriglyceridemia, which is either permanent orlimited to the postprandial phase.

Example 6 Effect of Leptin on the Expression of LSR; Therapeutic Effects

[0614] To reinforce correlation between the administration of leptin,the reduction in the postprandial lipemic response, and an enhancedexpression or activity of the LSR receptor, and to better evaluate thepossible therapeutic implications of the induction of the activity ofhepatic clearance of lipoproteins by leptin, the inventors supplementedthe preceding analysis with monitoring of the weight variation, of theLSR activity and of the expression of LSR mRNA, in control or obeseanimals treated with leptin or otherwise.

[0615] Postprandial Lipemic Response and LSR Activity in Control andObese Mice

[0616] Control male mice (C57BL6) (n=8) and obese male mice (ob/ob,n=8—animals deficient in the leptin gene—and db/db, n=8—animalsdeficient in the gene for the leptin receptor-) (aged 17 weeks old) wereweighed in order to quantitatively establish the differences in weightbetween lines (FIG. 28A). The postprandial lipemic responses of theanimals of each line were measured in the absence of treatment withleptin as described above. The apparent number of LSR receptorsexpressed at the surface of the hepatic cells was measured on 4 animalsof each line, as described above, and expressed in comparison with the5′-nucleotidase activity (enzyme selectively measured at the level ofthe plasma membranes; Sigma kit). Finally, Northern blotting made itpossible to estimate the level of expression of the LSR receptor inthree animals of each line, according to the protocol described above.

[0617] The higher postprandial lipemic response in the obese animals(FIG. 28B) is in agreement with the smaller apparent number of hepaticLSR receptors in these same animals (FIG. 28C). Furthermore, theNorthern blotting results (FIG. 28D) indicate that this reduction in theapparent number of LSR receptors in the obese animals is accompanied bya reduction in the level of expression of the said receptor in the sameanimals. The inventors have shown that indeed, a reduction in the numberof mRNA encoding the LSR receptor is observed in the obese mice ob/oband db/db.

[0618] The inventors also studied the effect of a long-term treatment ofa treatment with leptin on ob/ob mice (FIG. 29).

[0619] Effect of a long-term treatment with leptin on ob/ob mice

[0620] The ob/ob obese mice received a daily injection of either leptin,or of an equivalent volume of sterile PBS, for 30 days. The injecteddoses are 50 μg/animal from day 0 to day 4, 100 μg/animal from day 5 today 17, and 150 μg/animal from day 18 to day 30.

[0621] Several parameters indicated below are measured:

[0622] the weight (FIG. 29A): the change in weight is measured for 6animals, over the duration of the treatment;

[0623] the postprandial lipemic response (FIG. 29B): it is measuredaccording to the protocol detailed in Example 5 on three animals in eachgroup, on day 29;

[0624] the apparent number of LSR receptors (FIG. 29C): it is measuredaccording to the protocol detailed in Example 4 on three animals in eachgroup, on day 30;

[0625] the quantity of LSR mRNA (FIG. 23D): it is estimated by Northernblotting as indicated in the protocol of Example 2.

[0626] The inventors thus observed a very significant loss of weight inthe ob/ob obese mice treated over 30 days with leptin. Furthermore, thetreatment with leptin causes a clear reduction in the postprandiallipemic response. This reduction in the postprandial lipemic response iscorrelated with an increase in the apparent number of LSR receptors atthe surface of the cells and with an increase in the quantity of mRNAencoding the subunits of the LSR receptor.

[0627] These results establish in vivo that LSR represents the limitingstep in the elimination of dietary lipids. Furthermore, the treatment ofthis obesity inducing a weight loss causes an increase in the activityof hepatic degradation of dietary lipids, and a reduction in thepostprandial lipemic response.

Example 7 Characterization of the Human LSR Receptor

[0628] Northern-blot Analysis

[0629] Nucleic probes for rat LSR were used to carry out Northern-blotanalyses with a membrane (Human Multiple Tissue Northern Blot, Clontech#7760-1) comprising human heart, brain, placenta, lung, liver, skeletalmuscle, kidney and pancreas poly A RNAs. A band of about 2 kbp isdetected in the liver and in the kidney. Approximate quantification ofthe hybridization results indicate that LSR is expressed in the liver atleast 5 times more than in the kidney.

[0630] Cloning of the cDNA; Study of the Splicing Zone

[0631] Reverse transcription-PCR experiments on the mRNA made itpossible to determine with greater precision the size of exon 1 on the5′ side and splicing sites between exons 1 and 2. However, it is notcertain that this end constitutes the start of this exon. In addition, asecond initiation site exists in exon 1 which is more downstream fromthe first and which exhibits a greater probability than the latter. Thesplicing between exons 1 and 2 was different between the human RNA andthe rat RNA.

[0632] The amplification was carried out with several pairs of primers:a: 5′-ATGCAACAGGACGGACTTGGA-3′ (SEQ ID 31) exon 1 b:5′-TCAGACGACTAAACTTTCCCGACTCAGG-3′ (SEQ ID 32) exon 10 c:5′-CTACAACCCCTACGTTGAGT-3′ (SEQ ID 33) exon 2 d:5′-TCGTGACCTGACCTTTGACCAGAC-3′ (SEQ ID 34) exon 3 e:5′-CCTGAGCTACTCCTGTCAACGTCT-3′ (SEQ ID 35) exon 6 f:5′-AGGCCGAGATCGCCAGTCGT-3′ (SEQ ID 36) exon 9

[0633] The amplification carried out with the ab pair of primers led totwo products 1.8 kb and 2 kb in size after separation on anelectrophoresis gel. Given that the sizes of these two products can beexplained by an alternative splicing similar to that described in rats,the other amplification primers were drawn. These primers made itpossible to identify the three forms of cDNA resulting from thealternative splicing of the RNA.

[0634] The first cDNA which contains the totality of the ten exons iscalled LSR-Hs-2062 and corresponds to SEQ ID 7. It corresponds to therat cDNA LSR-Rn-2097. The second cDNA contains exons 1, 2, 3, 5, 6, 7,8, 9 and 10, and is called LSR-Hs-2005. It corresponds to SEQ ID 9. ThiscDNA corresponds to the rat cDNA LSR-Rn-2040. Finally, the cDNAcontaining exons 1, 2, 3, 6, 7, 8, 9 and 10 is called LSR-Hs-1858 andits sequence is listed in SEQ ID 11. It corresponds to the rat cDNALSR-Rn-1893.

[0635] It should be noted that it was possible to demonstrate a slippageof the splicing site at the boundary of exon 8. This slippage, of thetriplet TAG at position 19953-19955 of SEQ ID 19 to the contiguoustriplet AAG at position 19956-19958 of SEQ ID 19, results in the loss ofthe Glu residue at position 386 of the cDNA of SEQ ID 8.

[0636] The sequences of the proteins encoded by the cDNA LSR-Hs-2062,LSR-Hs-2005 and LSR-Hs-1858 correspond respectively to SEQ ID 8, 10 and12. The biological protein sequences can start at the first ATG codonobserved in the reading frame (position 35 of the protein sequence).However, the preferred codon for initiation of translation is moredownstream at position 83 of the protein sequence. Furthermore, it isquite possible that this initiation codon is more upstream in the 5′region of exon 1 not yet determined or in a possible exon preceding thelatter.

[0637] Finally, FIGS. 3A and 3B represents a schematic representation ofthe various protein forms identified in humans, indicating the conservedmotifs.

[0638] This analysis makes it possible to conclude that three α, α and βsubunits of LSR, which are equivalent to the LSR 66, LSR 64 and LSR 58forms in rats, exists in humans.

[0639] Identification and Isolation of the Genomic Sequence for HumanLSR

[0640] Screening of public data banks of nucleic sequences (Genebank,version: 101) both with the sequence of mouse lisch7 (Accession No.:U49507) and with that of rat LSR_(—)2097 isolated by the inventors madeit possible to isolate two human genomic DNA sequences. They are cosmidswhose accession numbers are AC002128 and AD000684, of respective sizes45,328 bp and 41,936 bp. These two cosmids partially overlap. The 3′ endof the cosmid AC002128 overlaps, over 12838 bp, the 5′ end of the cosmidAD000684. On the common portion of 12,838 bp, the sequences are 100%identical, apart from two deletions at positions 822 and 3170 of thecosmid AD000684. The human LSR gene is distributed over the two cosmids.To facilitate the study of this region, a complete genomic sequence wasreconstituted: the 45,328 bp of the cosmid AC002128 were added to thesequence of the cosmid AD000684 between the 12,839 base and the 41,936base. The combination constitutes a sequence of 74,426 bp. A genomicsequence covering the LSR gene, was extracted (SEQ ID 19).

[0641] The putative exons of the LSR gene were determined afteralignment of the sequence described above with the sequences of the RNAsfor mouse Lisch7 and rat LSR. The validity of the splicing sites oneither side of the putative exons was verified.

[0642] Moreover, a human genomic library consisting of BACs was screenedby the methods described in Chumakov et al., 1995; the clones thusisolated were contiged, subcloned and then sequenced in order to obtainthe human genomic sequence encoding LSR (SEQ ID 41).

[0643] The two sequences thus obtained (SEQ ID 19 and 41) carry minordifferences which are mentioned in the accompanying listings.

Example 8 LSR Activity in Humans

[0644] Primary cultures of human fibroblasts, isolated from subjectshaving a deletion affecting the promoter and the first exon of the LDLreceptor gene, were obtained.

[0645] The incubation of these cells in the presence and in the absenceof oleate shows that the latter induces LDL binding, internalization anddegradation activity which follows a saturation kinetics (Bihain andYen, 1992). The affinity of this receptor, induced by oleate, is maximumfor the particles high in triglycerides (VLDL and chylomicrons) as wellas for triolein and phosphatidylcholine supplemented with recombinantapoprotein E. The affinity of the LDLs for the receptor is lower thanthat of the VLDLs and the chylomicrons but, however, higher than thoseof triolein and phosphatidylcholine particles not containing ApoE, orthan those of VLDLs isolated from a subject with type III hyperlipidemiaand the ApoE E_(2/2) phenotype (Yen et al., 1994).

[0646] It was also possible to measure the LSR activity in fibroblastsof normal human subjects (FIG. 30), according to the protocol below.

[0647] Measurement of the Binding, Internalization and Degradation ofLDLs by Fibroblasts.

[0648] The fibroblasts are cultured beforehand for one week as describedabove, except that the medium contains 20% foetal bovine serum(Goldstein et al., 1983). Next, they are incubated with increasingconcentrations of ¹²⁵I-LDL in the absence or in the presence of 1 mMoleate. The cells are then washed, lysed and counted for theirradioactivity.

Example 9 Effect of Leptin on the LSR Activity in Humans

[0649] The LSR activity of human fibroblasts HF (familialhypercholesterolemia) is also increased after incubation with leptin(FIG. 31), suggesting that as in rats, LSR participates, in humans, inthe clearance of cytokines, and its activity is modulated by the latter.The corresponding measurements were carried out as indicated below.

[0650] Effect of Leptin on the LSR Activity on Human Fibroblasts

[0651] The fibroblasts HF are incubated for 30 minutes at 37° C. withincreasing concentrations of leptin, and then for 2 hours at 37° C. with50 μg/ml of ¹²⁵I-LDL, in the presence of 500 μM oleate. The binding,internalization and degradation of the LDLs are measured as indicated inExample 1.

Example 10 Cloning of the cDNA for Mouse LSR; Analysis of the Productsof Alternative Splicing

[0652] The cloning of the cDNA for mouse LSR was carried out using amouse liver mRNA library. The cloning method used is the same as thatfor the cDNA for human LSR. The mRNAs were purified and a reversetranscription PCR amplification was carried out with the specific DNAprimers. The amplification fragment was cloned to a TA cloning vector(Introgene).

[0653] A study of the products of alternative splicing with primerssituated in exon 2 and in exon 9 was also carried out in a mannersimilar to that carried out for the human LSR.

[0654] Three products of alternative splicing were observed:LSR-Mm-1886, LSR-Mm-1829 and LSR-Mm-1682. LSR-Mm-1886 contains all theexons from 1 to 10. LSR-Mm-1829 and LSR-Mm-1682 lack exon 4 and exons 4and 5, respectively. These three biological forms of cDNA indeedcorrespond to what was observed in humans and rats. The nucleotidesequences of the cDNAs LSR-Mm-1886, LSR-Mm-1829 and LSR-Mm-1682 areillustrated in SEQ ID 13, 14 and 15, respectively. The protein sequencesencoded by the cDNAs LSR-Mm-1886, LSR-Mm-1829 and LSR-Mm-1682 areillustrated in SEQ ID 16, 17 and 18.

Example 11 Identification of the γ Subunit of LSR

[0655] The α and β subunits of LSR were identified as indicated above.Analysis of the products of translation of the RNAs encoding these twosubunits does not allow the presence of a third subunit of molecularweight≈35 kDa to be explained. This subunit is detected only afterreduction of the LSR complex (FIG. 10, lane 4).

[0656] We purified and obtained the NH₂-terminal sequence of this γsubunit.

[0657] The purification was carried out by immunoaffinity chromatographyaccording to the following procedure.

[0658] Purification of the γ Subunit of LSR

[0659] Anti-LSR antibodies (band A) are coupled to a resin [2.5 mg ofIgG per 3.5 ml of affi-gel Hz immunoaffinity kit resin (Biorad153-6060)] which is then incubated with proteins solubilized from totalmembranes of rat liver (20 mM Tris buffer, 2 mM EDTA, 0.125 M octylglucoside (5×CMC), 1% inhibitor cocktail, pH=7.4: 160 mg of membraneproteins give 41.3 mg of solubilized proteins (SP) in a volume of 17 ml.

[0660] The incubation is carried out for 12 hours: 17 ml filled to 50 mlwith 20 mM Tris buffer, 2 mM EDTA, pH 7.4 and the 3.5 ml of resin, withrotary shaking, at room temperature. The resin is washed with 40 ml of20 mM Tris buffer, 2 mM EDTA, pH 7.4 and then eluted with 20 mM Trisbuffer, 2 mM EDTA, 200 mM glycine, pH 2.5 in 30 fractions of 500 μl. ThepH of each fraction is neutralized with 100 μl per tube of 1 M Trisbuffer, 2 mM EDTA, pH 9. 50 μl of each fraction are deposited on anitrocellulose membrane for dot-blot analysis: incubation with anti-LSRantibody, and then with a second antibody coupled to alkalinephosphatase.

[0661] The positive fractions from 7 to 28 are pooled in pairs andconcentrated 2.5-fold in a Speedvac. Western blotting is carried out onthe pooled, concentrated and separated fractions on a 10% PAGE-SDS gel.Bands are observed in fractions 7 to 14 (the fractions are pooled).

[0662] The two pools are dialysed against 24 mM ammonium bicarbonate andthen freeze-dried in a Speedvac. The powder is taken up in 80 μl of 20mM Tris buffer, 2 mM EDTA, 2% SDS, 3% urea, pH 7.4 and reduced in thepresence of 5% P-mercaptoethanol for 30 minutes at 100° C.

[0663] After migration and wet transfer in 50 mM Tris, 50 mM borate on asequencing membrane (PVDF) at 30 mA, the membrane is stained with amidoblack.

[0664] A band with an apparent MW of about 35 kDa was thus identifiedand sent for sequencing according to the Edman method.

[0665] The sequence obtained is LHTGDKAFVEFLTDEIKEE. This sequencecorresponds identically to that of a protein of molecular weight 33 kDaidentified above as a protein of the cellular surface which binds theglobular heads of C1q (gC1q-R) (Ghebrehiwet et al., 1994). A more recentobservation indicates that this potential receptor for C1q is alsolocated in the vesicles situated under the cellular surface (van denBerg et al., 1997). This protein also corresponds to a proteinpreviously identified as p34, and which combines with a lamin receptor.This receptor possesses a long NH₂-terminal segment oriented inwards inthe cell nucleus as well as 8 transmembrane domains. This receptor bindsto lamin in a manner which depends on the degree of phosphorylation.Finally, gC1q-R combines with “splicing factor 2” (Honore et al., 1993).The lamin receptor and “splicing factor 2” have in common thecharacteristic of containing a repeated sequence of serine and arginine(RSRS) situated at the level of the NH₂, terminal segment in the case ofthe lamin receptor and at the level of the carboxy-terminal segment inthe case of SF2.

[0666] It is remarkable to observe that both A-LSR and β-LSR exhibitrepeated segments high in serine and arginine (FIG. 1). Our hypothesisis that the Y-LSR protein represents a molecular chaperone whichcombines with the α and β subunits of LSR via their RSRS domain.

[0667] In order to verify this hypothesis, we obtained polyclonalantibodies directed against two synthetic peptides whose sequence wassituated at the carboxy- or NH₂-terminal end of the gC1q-R protein:

[0668] —NH₂-terminal peptide of gC1q-R: LRCVPRVLGSSVAGY* (amino acids 5to 19 of gC1qR) (SEQ ID 39)

[0669] —COOH-terminal peptide of gC1q-R: C*YITFLEDLKSFVKSQ (amino acids268 to 282 of gC1q-R) (SEQ ID 40).

[0670] *: amino acids differing from the protein sequence, so as tooptimize the antigenicity of the peptides.

[0671]FIG. 32 shows these antibodies specifically inhibit the activityof LSR. The antibody directed against the COOH-terminal end appears tobe the most effective. These results indicate that gC1q-R, or one of itsstructurally similar homologues, represents a molecular chaperonenoncovalently combined with the LSR multimeric complex.

Example 12 Regulation of the LSR Activity by C1q and its Homologues

[0672] It has been shown that gC1q-R could bind the globular head ofcomplement factor 1. We sought to use this property of C1q to displacegC1q-R combined with LSR, and we measured the effect of increasing dosesof C1q on the binding, internalization and degradation of the LDLs byhepatocytes in primary culture. FIG. 33 shows an increase in the captureand degradation of LDLs induced by human C1q, even in the absence ofoleate.

[0673] A less substantial, but nevertheless significant, increase isalso observed in the presence of oleate. However, under theseconditions, the maximum effect is obtained for lower concentrations ofC1q.

[0674] It therefore appears that gC1q-R exerts on LSR an inhibitoryeffect which is comparable to that induced by the 39 kD RAP for LRP, theLDL receptor and LSR (Troussard et al., 1995). The displacement of thechaperone gC1q-R using its capacity to bind to complement C1q makes itpossible to lift the inhibitory effect. Analysis of the gC1q-R sequenceshows that it may be a typical membrane receptor. Indeed, the proteinpossesses no hydrophobic sequence capable of crossing the phospholipidbilayer.

[0675] The effect of complement C1q on the activity of LSR opens majorperspectives in the context of the genetics of obesity. It is possible,indeed, that mutations affecting either the gene for C1q, that forgC1q-R, or alternatively that for their analogues such as for exampleAdipoQ, cerebellin, collagen alpha 1-10, SPA and SPD (pulmonarysurfactant proteins), mannan-binding protein, and the scavenger receptoror its homologue LRP (Hu et al., 1996; Drickamer et al., 1986; Kriegerand Herz, 1994; Elomaa et al., 1995) modulate the activity of LSR, bothas regards clearance of lipoproteins and as regards that of leptin.

[0676] Several proteins can interact with gC1q-R because they exhibithomologies with complement C1q. In particular, two proteins isolated inmice, AdipoQ (Hu et al., 1996) and acrp30 (Scherer et al., 1995), and ahuman protein APM1 (Maeda et al., 1996) exhibit marked homologies. Thesethree proteins, like the components of complement C1q (C1q A, B, C), aresecreted proteins; they have an NH₂-terminal end which resemblescollagen (repetition of Gly-X—Y motifs) and a COOH-terminal endcorresponding to the globular domain of complement C1q. These threeproteins are preferably expressed in the adipose tissue. There are only3 amino acids differing between AdipoQ and acrp30. APM1, a protein whosemessenger has been characterized as being highly expressed inadipocytes, exhibits 79.7% nucleic acid identity and 80.6% amino acididentity with AdipoQ. APM1 is therefore certainly the human homologue ofAdipoQ.

Example 13 Screening of Compounds Modifying the Activity of the LSRReceptor

[0677] As described above, the inventors formulated the hypothesis thatthe LSR “γ band ”, a protein which is highly homologous to gC1qR, mightinteract with the LSR receptor like a molecular chaperone and might thusform an “LSR complex ”, comprising the a or α and β subunits of the LSRreceptor and a gC1qR type molecule gC1qR has been previously identifiedas a cell surface protein which binds the globular heads of thecomplement factor C1q. In addition to C1q, several proteins exhibitinghomologies with the C1q proteins, in particular AdipoQ and acrp30 inmice and APM1 in humans, are capable of interacting with the proteinhomologous to gC1qR in the LSR complex and of modifying the LSRactivity.

[0678] Screening Parameters

[0679] The screening of a compound such as C1q or AdipoQ was carried outthrough the measurement of various parameters of which the mostimportant is the measurement of the effect of the compound on theactivity of the LSR receptor. The various parameters are the following:

[0680] change in weight

[0681] food intake

[0682] postprandial lipemic response

[0683] binding, internalization and/or degradation of lipoproteins suchas the LDLs.

[0684] Change in Weight

[0685] Osmotic pumps were surgically inserted into the abdominalcavities of 12 Sprague-Dawley male rats of 400-450 g. The osmotic pumpscontained either 2 ml of PBS (phosphate buffered saline), pH 7.4(control 6 rats), or 2 ml of recombinant AdipoQ protein (5 mg/ml PBS, 6rats). These pumps were designed to deliver 10 μl/h (50 μg AdipoQ/h).The animals are weighed and individually housed in metabolic cages. 3animals in each group are subjected ad libitum either to a normal dietor to a fatty diet (day 0). The fatty diet consists of a normal dietsupplemented with 2% (w/w) cholesterol, 10% (w/w) saturated fatty acidin the form of vegetaline, [lacuna] % (w/w) sunflower oil and 15% (w/w)sucrose. On day 3, the animals are weighed and blood samples areobtained from the caudal vein. The quantity of plasma triglycerides wasmeasured using an enzymatic kit.

[0686] Food Intake

[0687] Recombinant AdipoQ protein (100 μg) or PBS alone were injecteddaily for 5 days through the caudal vein of ob/ob or db/db mice kept ina metabolic cage. The mice are weighed each day and the quantity of foodconsumed was also measured. The results correspond to a mean food intakeand a standard deviation for 4 mice in each group.

[0688] Postprandial Lipemic Response

[0689] Male Sprague-Dawley rats (400-450 g), starved since the daybefore, were force-fed with a meal which was very high in fat (t=0) (60%fatty acid of which 37% saturated, 27% monounsaturated and 36%polyunsaturated, 20% protein and 20% carbohydrate, the total providing56 kcal/kg of body weight) and received immediately afterwards anintravenous injection (femoral vein) of either 300 μl of PBS alone or ofthe same volume containing 1 mg of mouse recombinant AdipoQ protein.Blood samples were collected at various times (0, 2, 4 and 6 h). Thequantity of plasma triglycerides was measured using an enzymatic kit.The results are presented as mean values and standard deviations on 3animals.

[0690] LSR Activity or Binding, Internalization and Degradation ofLipoproteins

[0691] Primary cultures of rat hepatocytes were prepared and distributedinto 6-well plates (9000,000 cells/well). After 48 h, the cells werewashed once with PBS (2 ml/well) and incubated for 30 min at 37° C. with20 ng/ml of recombinant murine leptin. The cells were then incubated for4 h at 37° C. with increasing concentrations of recombinant murineAdipoQ proteins and 20 μg/ml ¹²⁵I-LDL in the presence or in the absenceof 0.5 mM oleate. The binding, internalization and degradation oflipoproteins were measured as indicated in Example 1.

[0692] C1q

[0693] The compound C1q was tested for its capacity to modulate theactivity of the LSR receptor (binding, internalization and degradationof lipoproteins). FIG. 33 shows that the compound C1q exhibits theproperty of increasing the activity in the presence and in the absenceof oleate. Thus, it was possible for this compound C1q to be selected asmodulator of the LSR activity through the test of activity describedabove.

[0694] AdipoQ

[0695] The compound AdipoQ was tested according to the four parameterspresented above.

[0696]FIG. 34 shows that the compound AdipoQ modulates the LSR activityin the presence of oleate. Indeed, at the concentration of 25 ng/ml, itincreases the LSR activity.

[0697]FIG. 35 shows that the administration of AdipoQ makes it possibleto massively reduce the postprandial lipemic response.

[0698]FIG. 36 shows that a 3-day ip infusion treatment with AdipoQcauses a loss in weight which is much greater when the rat is subjectedto a fatty diet. Furthermore, the inventors observed that the level ofplasma triglycerides is reduced in the animals treated with AdipoQ.

[0699]FIG. 37 shows that an injection of AdipoQ reduces the food intakein obese animals.

[0700] The increase in the LSR activity induced by 25 ng/ml of AdipoQcan explain the reduction in the postprandial lipemic response and theweight loss.

[0701] Thus, the AdipoQ protein is a very valuable compound which couldbe used in particular in the treatment of obesity. The selection of thisprotein as a candidate molecule in the treatment of obesity validatesthe parameters for screening a compound of interest modulating the LSRactivity, the most important parameter consisting in measuring the LSRactivity.

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1 41 1 2097 DNA Rattus norvegicus 1 accgctcacc aggtcagttg tccccggaaagccgaaggca tgagcttcgc ccaagttctt 60 tttatgggtt agaactcctc cagagcgggggaaaaaggac ttggaatagg ggcgggacgg 120 agcacgcacc cttctccgcc ttggttctcgccgcgccccc tactctcggg atacttggga 180 ggggacgcgc gggcaccgtc gctgctagacggccgcg atg gcg ccg gcg gcc ggc 235 Met Ala Pro Ala Ala Gly 1 5 gcg tgtgct ggg gcg cct gac tcc cac cca gct acc gtg gtc ttc gtg 283 Ala Cys AlaGly Ala Pro Asp Ser His Pro Ala Thr Val Val Phe Val 10 15 20 tgt ctc tttctc atc att ttc tgc cca gac cct gcc agt gcc atc cag 331 Cys Leu Phe LeuIle Ile Phe Cys Pro Asp Pro Ala Ser Ala Ile Gln 25 30 35 gtg act gtg tctgac ccc tac cac gta gtg atc ctg ttc cag cca gtg 379 Val Thr Val Ser AspPro Tyr His Val Val Ile Leu Phe Gln Pro Val 40 45 50 acc ctg ccc tgc acctat cag atg agc aac act ctc aca gtc ccc atc 427 Thr Leu Pro Cys Thr TyrGln Met Ser Asn Thr Leu Thr Val Pro Ile 55 60 65 70 gtg atc tgg aag tacaag tca ttc tgc cgg gac cgt att gcc gat gcc 475 Val Ile Trp Lys Tyr LysSer Phe Cys Arg Asp Arg Ile Ala Asp Ala 75 80 85 ttc tct cct gcc agt gtggac aac cag cta aat gcc cag ttg gca gct 523 Phe Ser Pro Ala Ser Val AspAsn Gln Leu Asn Ala Gln Leu Ala Ala 90 95 100 ggc aac ccc ggc tac aacccc tat gtg gag tgc cag gac agt gta cgc 571 Gly Asn Pro Gly Tyr Asn ProTyr Val Glu Cys Gln Asp Ser Val Arg 105 110 115 act gtc agg gtg gtg gccacc aaa cag ggc aat gcg gtg acc ctg gga 619 Thr Val Arg Val Val Ala ThrLys Gln Gly Asn Ala Val Thr Leu Gly 120 125 130 gac tac tac caa ggc aggagg atc acc ata aca gga aat gct gac ctg 667 Asp Tyr Tyr Gln Gly Arg ArgIle Thr Ile Thr Gly Asn Ala Asp Leu 135 140 145 150 acc ttc gag cag acagcc tgg gga gac agt gga gtg tat tac tgc tct 715 Thr Phe Glu Gln Thr AlaTrp Gly Asp Ser Gly Val Tyr Tyr Cys Ser 155 160 165 gtg gtc tcg gcc caagat ctg gat gga aac aac gag gcg tac gca gag 763 Val Val Ser Ala Gln AspLeu Asp Gly Asn Asn Glu Ala Tyr Ala Glu 170 175 180 ctc atc gtc ctt ggcagg acc tca gag gcc cct gag ctc cta cct ggt 811 Leu Ile Val Leu Gly ArgThr Ser Glu Ala Pro Glu Leu Leu Pro Gly 185 190 195 ttt cgg gcg ggg cccttg gaa gat tgg ctc ttt gtg gtc gtg gtc tgc 859 Phe Arg Ala Gly Pro LeuGlu Asp Trp Leu Phe Val Val Val Val Cys 200 205 210 ctg gcg agc ctc ctcctc ttc ctc ctc ctg ggc atc tgc tgg tgc cag 907 Leu Ala Ser Leu Leu LeuPhe Leu Leu Leu Gly Ile Cys Trp Cys Gln 215 220 225 230 tgc tgt cct cacacc tgc tgc tgc tat gtc cga tgt ccc tgc tgc cca 955 Cys Cys Pro His ThrCys Cys Cys Tyr Val Arg Cys Pro Cys Cys Pro 235 240 245 gac aag tgc tgttgc cct gag gct ctt tat gct gct ggc aaa gca gcc 1003 Asp Lys Cys Cys CysPro Glu Ala Leu Tyr Ala Ala Gly Lys Ala Ala 250 255 260 acc tca ggt gtcccg agc atc tat gcc ccc agc atc tat acc cac ctc 1051 Thr Ser Gly Val ProSer Ile Tyr Ala Pro Ser Ile Tyr Thr His Leu 265 270 275 tca cct gcc aagacc cca cca cct ccg cct gcc atg att ccc atg ggc 1099 Ser Pro Ala Lys ThrPro Pro Pro Pro Pro Ala Met Ile Pro Met Gly 280 285 290 cct ccc tat gggtac cct gga gac ttt gac aga cat agc tca gtt ggt 1147 Pro Pro Tyr Gly TyrPro Gly Asp Phe Asp Arg His Ser Ser Val Gly 295 300 305 310 ggc cac agctcc caa gta ccc ctg ctg cgt gac gtg gat ggc agt gta 1195 Gly His Ser SerGln Val Pro Leu Leu Arg Asp Val Asp Gly Ser Val 315 320 325 tct tca gaagta cga agt ggc tac agg atc cag gct aac cag caa gat 1243 Ser Ser Glu ValArg Ser Gly Tyr Arg Ile Gln Ala Asn Gln Gln Asp 330 335 340 gac tcc atgagg gtc cta tac tat atg gag aaa gag cta gcc aac ttt 1291 Asp Ser Met ArgVal Leu Tyr Tyr Met Glu Lys Glu Leu Ala Asn Phe 345 350 355 gac cct tcccga cct ggc cct ccc aat ggc aga gtg gaa cgg gcc atg 1339 Asp Pro Ser ArgPro Gly Pro Pro Asn Gly Arg Val Glu Arg Ala Met 360 365 370 agt gaa gtaacc tcc ctc cat gaa gat gac tgg cga tcg agg cct tcc 1387 Ser Glu Val ThrSer Leu His Glu Asp Asp Trp Arg Ser Arg Pro Ser 375 380 385 390 agg gctcct gcc ctc acc ccc atc agg gat gag gag tgg aat cgc cac 1435 Arg Ala ProAla Leu Thr Pro Ile Arg Asp Glu Glu Trp Asn Arg His 395 400 405 tcc ccacag agt ccc aga aca tgg gag cag gaa ccc ctt caa gaa caa 1483 Ser Pro GlnSer Pro Arg Thr Trp Glu Gln Glu Pro Leu Gln Glu Gln 410 415 420 cca aggggt ggt tgg ggg tct gga cgc cct cgg gcc cgc tct gtg gat 1531 Pro Arg GlyGly Trp Gly Ser Gly Arg Pro Arg Ala Arg Ser Val Asp 425 430 435 gct ctagat gat atc aac cgg cct ggc tcc act gaa tca gga cgg tct 1579 Ala Leu AspAsp Ile Asn Arg Pro Gly Ser Thr Glu Ser Gly Arg Ser 440 445 450 tct ccccca agt agt gga cgg aga gga cgg gcc tat gca cct cca aga 1627 Ser Pro ProSer Ser Gly Arg Arg Gly Arg Ala Tyr Ala Pro Pro Arg 455 460 465 470 agtcgc agc cgg gat gac ctc tat gac ccg gac gat cct agg gac ttg 1675 Ser ArgSer Arg Asp Asp Leu Tyr Asp Pro Asp Asp Pro Arg Asp Leu 475 480 485 ccacat tcc cga gat ccc cac tat tat gac gac atc agg tct aga gat 1723 Pro HisSer Arg Asp Pro His Tyr Tyr Asp Asp Ile Arg Ser Arg Asp 490 495 500 ccacgt gct gac ccc aga tcc cgt cag cga tcc cga gat cct cgg gat 1771 Pro ArgAla Asp Pro Arg Ser Arg Gln Arg Ser Arg Asp Pro Arg Asp 505 510 515 gctggc ttc agg tca agg gac cct cag tat gat ggg cga cta tta gaa 1819 Ala GlyPhe Arg Ser Arg Asp Pro Gln Tyr Asp Gly Arg Leu Leu Glu 520 525 530 gaggct tta aag aaa aag ggg tcg ggc gag aga agg agg gtt tac agg 1867 Glu AlaLeu Lys Lys Lys Gly Ser Gly Glu Arg Arg Arg Val Tyr Arg 535 540 545 550gag gaa gaa gag gaa gag gag ggc caa tac ccc cca gca cct cca cct 1915 GluGlu Glu Glu Glu Glu Glu Gly Gln Tyr Pro Pro Ala Pro Pro Pro 555 560 565tac tca gag act gac tcg cag gcc tca cgg gag agg agg ctg aaa aag 1963 TyrSer Glu Thr Asp Ser Gln Ala Ser Arg Glu Arg Arg Leu Lys Lys 570 575 580aat ttg gcc ctg agt cgg gaa agt tta gtc gtc tga tccacgtttt 2009 Asn LeuAla Leu Ser Arg Glu Ser Leu Val Val * 585 590 gtatgtagct tttgtactttttttttaatt ggaatcaata ttgatgaaac ttcaagccta 2069 ataaaatgtc taatcacaaaaaaaaaaa 2097 2 593 PRT Rattus norvegicus 2 Met Ala Pro Ala Ala Gly AlaCys Ala Gly Ala Pro Asp Ser His Pro 1 5 10 15 Ala Thr Val Val Phe ValCys Leu Phe Leu Ile Ile Phe Cys Pro Asp 20 25 30 Pro Ala Ser Ala Ile GlnVal Thr Val Ser Asp Pro Tyr His Val Val 35 40 45 Ile Leu Phe Gln Pro ValThr Leu Pro Cys Thr Tyr Gln Met Ser Asn 50 55 60 Thr Leu Thr Val Pro IleVal Ile Trp Lys Tyr Lys Ser Phe Cys Arg 65 70 75 80 Asp Arg Ile Ala AspAla Phe Ser Pro Ala Ser Val Asp Asn Gln Leu 85 90 95 Asn Ala Gln Leu AlaAla Gly Asn Pro Gly Tyr Asn Pro Tyr Val Glu 100 105 110 Cys Gln Asp SerVal Arg Thr Val Arg Val Val Ala Thr Lys Gln Gly 115 120 125 Asn Ala ValThr Leu Gly Asp Tyr Tyr Gln Gly Arg Arg Ile Thr Ile 130 135 140 Thr GlyAsn Ala Asp Leu Thr Phe Glu Gln Thr Ala Trp Gly Asp Ser 145 150 155 160Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gln Asp Leu Asp Gly Asn 165 170175 Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Gly Arg Thr Ser Glu Ala 180185 190 Pro Glu Leu Leu Pro Gly Phe Arg Ala Gly Pro Leu Glu Asp Trp Leu195 200 205 Phe Val Val Val Val Cys Leu Ala Ser Leu Leu Leu Phe Leu LeuLeu 210 215 220 Gly Ile Cys Trp Cys Gln Cys Cys Pro His Thr Cys Cys CysTyr Val 225 230 235 240 Arg Cys Pro Cys Cys Pro Asp Lys Cys Cys Cys ProGlu Ala Leu Tyr 245 250 255 Ala Ala Gly Lys Ala Ala Thr Ser Gly Val ProSer Ile Tyr Ala Pro 260 265 270 Ser Ile Tyr Thr His Leu Ser Pro Ala LysThr Pro Pro Pro Pro Pro 275 280 285 Ala Met Ile Pro Met Gly Pro Pro TyrGly Tyr Pro Gly Asp Phe Asp 290 295 300 Arg His Ser Ser Val Gly Gly HisSer Ser Gln Val Pro Leu Leu Arg 305 310 315 320 Asp Val Asp Gly Ser ValSer Ser Glu Val Arg Ser Gly Tyr Arg Ile 325 330 335 Gln Ala Asn Gln GlnAsp Asp Ser Met Arg Val Leu Tyr Tyr Met Glu 340 345 350 Lys Glu Leu AlaAsn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly 355 360 365 Arg Val GluArg Ala Met Ser Glu Val Thr Ser Leu His Glu Asp Asp 370 375 380 Trp ArgSer Arg Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp 385 390 395 400Glu Glu Trp Asn Arg His Ser Pro Gln Ser Pro Arg Thr Trp Glu Gln 405 410415 Glu Pro Leu Gln Glu Gln Pro Arg Gly Gly Trp Gly Ser Gly Arg Pro 420425 430 Arg Ala Arg Ser Val Asp Ala Leu Asp Asp Ile Asn Arg Pro Gly Ser435 440 445 Thr Glu Ser Gly Arg Ser Ser Pro Pro Ser Ser Gly Arg Arg GlyArg 450 455 460 Ala Tyr Ala Pro Pro Arg Ser Arg Ser Arg Asp Asp Leu TyrAsp Pro 465 470 475 480 Asp Asp Pro Arg Asp Leu Pro His Ser Arg Asp ProHis Tyr Tyr Asp 485 490 495 Asp Ile Arg Ser Arg Asp Pro Arg Ala Asp ProArg Ser Arg Gln Arg 500 505 510 Ser Arg Asp Pro Arg Asp Ala Gly Phe ArgSer Arg Asp Pro Gln Tyr 515 520 525 Asp Gly Arg Leu Leu Glu Glu Ala LeuLys Lys Lys Gly Ser Gly Glu 530 535 540 Arg Arg Arg Val Tyr Arg Glu GluGlu Glu Glu Glu Glu Gly Gln Tyr 545 550 555 560 Pro Pro Ala Pro Pro ProTyr Ser Glu Thr Asp Ser Gln Ala Ser Arg 565 570 575 Glu Arg Arg Leu LysLys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val 580 585 590 Val 3 2040 DNARattus norvegicus 3 accgctcacc aggtcagttg tccccggaaa gccgaaggcatgagcttcgc ccaagttctt 60 tttatgggtt agaactcctc cagagcgggg gaaaaaggacttggaatagg ggcgggacgg 120 agcacgcacc cttctccgcc ttggttctcg ccgcgccccctactctcggg atacttggga 180 ggggacgcgc gggcaccgtc gctgctagac ggccgcg atggcg ccg gcg gcc ggc 235 Met Ala Pro Ala Ala Gly 1 5 gcg tgt gct ggg gcgcct gac tcc cac cca gct acc gtg gtc ttc gtg 283 Ala Cys Ala Gly Ala ProAsp Ser His Pro Ala Thr Val Val Phe Val 10 15 20 tgt ctc ttt ctc atc attttc tgc cca gac cct gcc agt gcc atc cag 331 Cys Leu Phe Leu Ile Ile PheCys Pro Asp Pro Ala Ser Ala Ile Gln 25 30 35 gtg act gtg tct gac ccc taccac gta gtg atc ctg ttc cag cca gtg 379 Val Thr Val Ser Asp Pro Tyr HisVal Val Ile Leu Phe Gln Pro Val 40 45 50 acc ctg ccc tgc acc tat cag atgagc aac act ctc aca gtc ccc atc 427 Thr Leu Pro Cys Thr Tyr Gln Met SerAsn Thr Leu Thr Val Pro Ile 55 60 65 70 gtg atc tgg aag tac aag tca ttctgc cgg gac cgt att gcc gat gcc 475 Val Ile Trp Lys Tyr Lys Ser Phe CysArg Asp Arg Ile Ala Asp Ala 75 80 85 ttc tct cct gcc agt gtg gac aac cagcta aat gcc cag ttg gca gct 523 Phe Ser Pro Ala Ser Val Asp Asn Gln LeuAsn Ala Gln Leu Ala Ala 90 95 100 ggc aac ccc ggc tac aac ccc tat gtggag tgc cag gac agt gta cgc 571 Gly Asn Pro Gly Tyr Asn Pro Tyr Val GluCys Gln Asp Ser Val Arg 105 110 115 act gtc agg gtg gtg gcc acc aaa cagggc aat gcg gtg acc ctg gga 619 Thr Val Arg Val Val Ala Thr Lys Gln GlyAsn Ala Val Thr Leu Gly 120 125 130 gac tac tac caa ggc agg agg atc accata aca gga aat gct gac ctg 667 Asp Tyr Tyr Gln Gly Arg Arg Ile Thr IleThr Gly Asn Ala Asp Leu 135 140 145 150 acc ttc gag cag aca gcc tgg ggagac agt gga gtg tat tac tgc tct 715 Thr Phe Glu Gln Thr Ala Trp Gly AspSer Gly Val Tyr Tyr Cys Ser 155 160 165 gtg gtc tcg gcc caa gat ctg gatgga aac aac gag gcg tac gca gag 763 Val Val Ser Ala Gln Asp Leu Asp GlyAsn Asn Glu Ala Tyr Ala Glu 170 175 180 ctc atc gtc ctt gat tgg ctc tttgtg gtc gtg gtc tgc ctg gcg agc 811 Leu Ile Val Leu Asp Trp Leu Phe ValVal Val Val Cys Leu Ala Ser 185 190 195 ctc ctc ctc ttc ctc ctc ctg ggcatc tgc tgg tgc cag tgc tgt cct 859 Leu Leu Leu Phe Leu Leu Leu Gly IleCys Trp Cys Gln Cys Cys Pro 200 205 210 cac acc tgc tgc tgc tat gtc cgatgt ccc tgc tgc cca gac aag tgc 907 His Thr Cys Cys Cys Tyr Val Arg CysPro Cys Cys Pro Asp Lys Cys 215 220 225 230 tgt tgc cct gag gct ctt tatgct gct ggc aaa gca gcc acc tca ggt 955 Cys Cys Pro Glu Ala Leu Tyr AlaAla Gly Lys Ala Ala Thr Ser Gly 235 240 245 gtc ccg agc atc tat gcc cccagc atc tat acc cac ctc tca cct gcc 1003 Val Pro Ser Ile Tyr Ala Pro SerIle Tyr Thr His Leu Ser Pro Ala 250 255 260 aag acc cca cca cct ccg cctgcc atg att ccc atg ggc cct ccc tat 1051 Lys Thr Pro Pro Pro Pro Pro AlaMet Ile Pro Met Gly Pro Pro Tyr 265 270 275 ggg tac cct gga gac ttt gacaga cat agc tca gtt ggt ggc cac agc 1099 Gly Tyr Pro Gly Asp Phe Asp ArgHis Ser Ser Val Gly Gly His Ser 280 285 290 tcc caa gta ccc ctg ctg cgtgac gtg gat ggc agt gta tct tca gaa 1147 Ser Gln Val Pro Leu Leu Arg AspVal Asp Gly Ser Val Ser Ser Glu 295 300 305 310 gta cga agt ggc tac aggatc cag gct aac cag caa gat gac tcc atg 1195 Val Arg Ser Gly Tyr Arg IleGln Ala Asn Gln Gln Asp Asp Ser Met 315 320 325 agg gtc cta tac tat atggag aaa gag cta gcc aac ttt gac cct tcc 1243 Arg Val Leu Tyr Tyr Met GluLys Glu Leu Ala Asn Phe Asp Pro Ser 330 335 340 cga cct ggc cct ccc aatggc aga gtg gaa cgg gcc atg agt gaa gta 1291 Arg Pro Gly Pro Pro Asn GlyArg Val Glu Arg Ala Met Ser Glu Val 345 350 355 acc tcc ctc cat gaa gatgac tgg cga tcg agg cct tcc agg gct cct 1339 Thr Ser Leu His Glu Asp AspTrp Arg Ser Arg Pro Ser Arg Ala Pro 360 365 370 gcc ctc acc ccc atc agggat gag gag tgg aat cgc cac tcc cca cag 1387 Ala Leu Thr Pro Ile Arg AspGlu Glu Trp Asn Arg His Ser Pro Gln 375 380 385 390 agt ccc aga aca tgggag cag gaa ccc ctt caa gaa caa cca agg ggt 1435 Ser Pro Arg Thr Trp GluGln Glu Pro Leu Gln Glu Gln Pro Arg Gly 395 400 405 ggt tgg ggg tct ggacgc cct cgg gcc cgc tct gtg gat gct cta gat 1483 Gly Trp Gly Ser Gly ArgPro Arg Ala Arg Ser Val Asp Ala Leu Asp 410 415 420 gat atc aac cgg cctggc tcc act gaa tca gga cgg tct tct ccc cca 1531 Asp Ile Asn Arg Pro GlySer Thr Glu Ser Gly Arg Ser Ser Pro Pro 425 430 435 agt agt gga cgg agagga cgg gcc tat gca cct cca aga agt cgc agc 1579 Ser Ser Gly Arg Arg GlyArg Ala Tyr Ala Pro Pro Arg Ser Arg Ser 440 445 450 cgg gat gac ctc tatgac ccg gac gat cct agg gac ttg cca cat tcc 1627 Arg Asp Asp Leu Tyr AspPro Asp Asp Pro Arg Asp Leu Pro His Ser 455 460 465 470 cga gat ccc cactat tat gac gac atc agg tct aga gat cca cgt gct 1675 Arg Asp Pro His TyrTyr Asp Asp Ile Arg Ser Arg Asp Pro Arg Ala 475 480 485 gac ccc aga tcccgt cag cga tcc cga gat cct cgg gat gct ggc ttc 1723 Asp Pro Arg Ser ArgGln Arg Ser Arg Asp Pro Arg Asp Ala Gly Phe 490 495 500 agg tca agg gaccct cag tat gat ggg cga cta tta gaa gag gct tta 1771 Arg Ser Arg Asp ProGln Tyr Asp Gly Arg Leu Leu Glu Glu Ala Leu 505 510 515 aag aaa aag gggtcg ggc gag aga agg agg gtt tac agg gag gaa gaa 1819 Lys Lys Lys Gly SerGly Glu Arg Arg Arg Val Tyr Arg Glu Glu Glu 520 525 530 gag gaa gag gagggc caa tac ccc cca gca cct cca cct tac tca gag 1867 Glu Glu Glu Glu GlyGln Tyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu 535 540 545 550 act gac tcgcag gcc tca cgg gag agg agg ctg aaa aag aat ttg gcc 1915 Thr Asp Ser GlnAla Ser Arg Glu Arg Arg Leu Lys Lys Asn Leu Ala 555 560 565 ctg agt cgggaa agt tta gtc gtc tga tccacgtttt gtatgtagct 1962 Leu Ser Arg Glu SerLeu Val Val * 570 tttgtacttt ttttttaatt ggaatcaata ttgatgaaac ttcaagcctaataaaatgtc 2022 taatcacaaa aaaaaaaa 2040 4 574 PRT Rattus norvegicus 4Met Ala Pro Ala Ala Gly Ala Cys Ala Gly Ala Pro Asp Ser His Pro 1 5 1015 Ala Thr Val Val Phe Val Cys Leu Phe Leu Ile Ile Phe Cys Pro Asp 20 2530 Pro Ala Ser Ala Ile Gln Val Thr Val Ser Asp Pro Tyr His Val Val 35 4045 Ile Leu Phe Gln Pro Val Thr Leu Pro Cys Thr Tyr Gln Met Ser Asn 50 5560 Thr Leu Thr Val Pro Ile Val Ile Trp Lys Tyr Lys Ser Phe Cys Arg 65 7075 80 Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gln Leu 8590 95 Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val Glu100 105 110 Cys Gln Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys GlnGly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly Arg Arg IleThr Ile 130 135 140 Thr Gly Asn Ala Asp Leu Thr Phe Glu Gln Thr Ala TrpGly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Ser Ala GlnAsp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr Ala Glu Leu Ile Val LeuAsp Trp Leu Phe Val Val 180 185 190 Val Val Cys Leu Ala Ser Leu Leu LeuPhe Leu Leu Leu Gly Ile Cys 195 200 205 Trp Cys Gln Cys Cys Pro His ThrCys Cys Cys Tyr Val Arg Cys Pro 210 215 220 Cys Cys Pro Asp Lys Cys CysCys Pro Glu Ala Leu Tyr Ala Ala Gly 225 230 235 240 Lys Ala Ala Thr SerGly Val Pro Ser Ile Tyr Ala Pro Ser Ile Tyr 245 250 255 Thr His Leu SerPro Ala Lys Thr Pro Pro Pro Pro Pro Ala Met Ile 260 265 270 Pro Met GlyPro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp Arg His Ser 275 280 285 Ser ValGly Gly His Ser Ser Gln Val Pro Leu Leu Arg Asp Val Asp 290 295 300 GlySer Val Ser Ser Glu Val Arg Ser Gly Tyr Arg Ile Gln Ala Asn 305 310 315320 Gln Gln Asp Asp Ser Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu 325330 335 Ala Asn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Val Glu340 345 350 Arg Ala Met Ser Glu Val Thr Ser Leu His Glu Asp Asp Trp ArgSer 355 360 365 Arg Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp GluGlu Trp 370 375 380 Asn Arg His Ser Pro Gln Ser Pro Arg Thr Trp Glu GlnGlu Pro Leu 385 390 395 400 Gln Glu Gln Pro Arg Gly Gly Trp Gly Ser GlyArg Pro Arg Ala Arg 405 410 415 Ser Val Asp Ala Leu Asp Asp Ile Asn ArgPro Gly Ser Thr Glu Ser 420 425 430 Gly Arg Ser Ser Pro Pro Ser Ser GlyArg Arg Gly Arg Ala Tyr Ala 435 440 445 Pro Pro Arg Ser Arg Ser Arg AspAsp Leu Tyr Asp Pro Asp Asp Pro 450 455 460 Arg Asp Leu Pro His Ser ArgAsp Pro His Tyr Tyr Asp Asp Ile Arg 465 470 475 480 Ser Arg Asp Pro ArgAla Asp Pro Arg Ser Arg Gln Arg Ser Arg Asp 485 490 495 Pro Arg Asp AlaGly Phe Arg Ser Arg Asp Pro Gln Tyr Asp Gly Arg 500 505 510 Leu Leu GluGlu Ala Leu Lys Lys Lys Gly Ser Gly Glu Arg Arg Arg 515 520 525 Val TyrArg Glu Glu Glu Glu Glu Glu Glu Gly Gln Tyr Pro Pro Ala 530 535 540 ProPro Pro Tyr Ser Glu Thr Asp Ser Gln Ala Ser Arg Glu Arg Arg 545 550 555560 Leu Lys Lys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val Val 565 570 51893 DNA Rattus norvegicus 5 accgctcacc aggtcagttg tccccggaaa gccgaaggcatgagcttcgc ccaagttctt 60 tttatgggtt agaactcctc cagagcgggg gaaaaaggacttggaatagg ggcgggacgg 120 agcacgcacc cttctccgcc ttggttctcg ccgcgccccctactctcggg atacttggga 180 ggggacgcgc gggcaccgtc gctgctagac ggccgcg atggcg ccg gcg gcc ggc 235 Met Ala Pro Ala Ala Gly 1 5 gcg tgt gct ggg gcgcct gac tcc cac cca gct acc gtg gtc ttc gtg 283 Ala Cys Ala Gly Ala ProAsp Ser His Pro Ala Thr Val Val Phe Val 10 15 20 tgt ctc ttt ctc atc attttc tgc cca gac cct gcc agt gcc atc cag 331 Cys Leu Phe Leu Ile Ile PheCys Pro Asp Pro Ala Ser Ala Ile Gln 25 30 35 gtg act gtg tct gac ccc taccac gta gtg atc ctg ttc cag cca gtg 379 Val Thr Val Ser Asp Pro Tyr HisVal Val Ile Leu Phe Gln Pro Val 40 45 50 acc ctg ccc tgc acc tat cag atgagc aac act ctc aca gtc ccc atc 427 Thr Leu Pro Cys Thr Tyr Gln Met SerAsn Thr Leu Thr Val Pro Ile 55 60 65 70 gtg atc tgg aag tac aag tca ttctgc cgg gac cgt att gcc gat gcc 475 Val Ile Trp Lys Tyr Lys Ser Phe CysArg Asp Arg Ile Ala Asp Ala 75 80 85 ttc tct cct gcc agt gtg gac aac cagcta aat gcc cag ttg gca gct 523 Phe Ser Pro Ala Ser Val Asp Asn Gln LeuAsn Ala Gln Leu Ala Ala 90 95 100 ggc aac ccc ggc tac aac ccc tat gtggag tgc cag gac agt gta cgc 571 Gly Asn Pro Gly Tyr Asn Pro Tyr Val GluCys Gln Asp Ser Val Arg 105 110 115 act gtc agg gtg gtg gcc acc aaa cagggc aat gcg gtg acc ctg gga 619 Thr Val Arg Val Val Ala Thr Lys Gln GlyAsn Ala Val Thr Leu Gly 120 125 130 gac tac tac caa ggc agg agg atc accata aca gga aat gct gac ctg 667 Asp Tyr Tyr Gln Gly Arg Arg Ile Thr IleThr Gly Asn Ala Asp Leu 135 140 145 150 acc ttc gag cag aca gcc tgg ggagac agt gga gtg tat tac tgc tct 715 Thr Phe Glu Gln Thr Ala Trp Gly AspSer Gly Val Tyr Tyr Cys Ser 155 160 165 gtg gtc tcg gcc caa gat ctg gatgga aac aac gag gcg tac gca gag 763 Val Val Ser Ala Gln Asp Leu Asp GlyAsn Asn Glu Ala Tyr Ala Glu 170 175 180 ctc atc gtc ctt gtt tat gct gctggc aaa gca gcc acc tca ggt gtc 811 Leu Ile Val Leu Val Tyr Ala Ala GlyLys Ala Ala Thr Ser Gly Val 185 190 195 ccg agc atc tat gcc ccc agc atctat acc cac ctc tca cct gcc aag 859 Pro Ser Ile Tyr Ala Pro Ser Ile TyrThr His Leu Ser Pro Ala Lys 200 205 210 acc cca cca cct ccg cct gcc atgatt ccc atg ggc cct ccc tat ggg 907 Thr Pro Pro Pro Pro Pro Ala Met IlePro Met Gly Pro Pro Tyr Gly 215 220 225 230 tac cct gga gac ttt gac agacat agc tca gtt ggt ggc cac agc tcc 955 Tyr Pro Gly Asp Phe Asp Arg HisSer Ser Val Gly Gly His Ser Ser 235 240 245 caa gta ccc ctg ctg cgt gacgtg gat ggc agt gta tct tca gaa gta 1003 Gln Val Pro Leu Leu Arg Asp ValAsp Gly Ser Val Ser Ser Glu Val 250 255 260 cga agt ggc tac agg atc caggct aac cag caa gat gac tcc atg agg 1051 Arg Ser Gly Tyr Arg Ile Gln AlaAsn Gln Gln Asp Asp Ser Met Arg 265 270 275 gtc cta tac tat atg gag aaagag cta gcc aac ttt gac cct tcc cga 1099 Val Leu Tyr Tyr Met Glu Lys GluLeu Ala Asn Phe Asp Pro Ser Arg 280 285 290 cct ggc cct ccc aat ggc agagtg gaa cgg gcc atg agt gaa gta acc 1147 Pro Gly Pro Pro Asn Gly Arg ValGlu Arg Ala Met Ser Glu Val Thr 295 300 305 310 tcc ctc cat gaa gat gactgg cga tcg agg cct tcc agg gct cct gcc 1195 Ser Leu His Glu Asp Asp TrpArg Ser Arg Pro Ser Arg Ala Pro Ala 315 320 325 ctc acc ccc atc agg gatgag gag tgg aat cgc cac tcc cca cag agt 1243 Leu Thr Pro Ile Arg Asp GluGlu Trp Asn Arg His Ser Pro Gln Ser 330 335 340 ccc aga aca tgg gag caggaa ccc ctt caa gaa caa cca agg ggt ggt 1291 Pro Arg Thr Trp Glu Gln GluPro Leu Gln Glu Gln Pro Arg Gly Gly 345 350 355 tgg ggg tct gga cgc cctcgg gcc cgc tct gtg gat gct cta gat gat 1339 Trp Gly Ser Gly Arg Pro ArgAla Arg Ser Val Asp Ala Leu Asp Asp 360 365 370 atc aac cgg cct ggc tccact gaa tca gga cgg tct tct ccc cca agt 1387 Ile Asn Arg Pro Gly Ser ThrGlu Ser Gly Arg Ser Ser Pro Pro Ser 375 380 385 390 agt gga cgg aga ggacgg gcc tat gca cct cca aga agt cgc agc cgg 1435 Ser Gly Arg Arg Gly ArgAla Tyr Ala Pro Pro Arg Ser Arg Ser Arg 395 400 405 gat gac ctc tat gacccg gac gat cct agg gac ttg cca cat tcc cga 1483 Asp Asp Leu Tyr Asp ProAsp Asp Pro Arg Asp Leu Pro His Ser Arg 410 415 420 gat ccc cac tat tatgac gac atc agg tct aga gat cca cgt gct gac 1531 Asp Pro His Tyr Tyr AspAsp Ile Arg Ser Arg Asp Pro Arg Ala Asp 425 430 435 ccc aga tcc cgt cagcga tcc cga gat cct cgg gat gct ggc ttc agg 1579 Pro Arg Ser Arg Gln ArgSer Arg Asp Pro Arg Asp Ala Gly Phe Arg 440 445 450 tca agg gac cct cagtat gat ggg cga cta tta gaa gag gct tta aag 1627 Ser Arg Asp Pro Gln TyrAsp Gly Arg Leu Leu Glu Glu Ala Leu Lys 455 460 465 470 aaa aag ggg tcgggc gag aga agg agg gtt tac agg gag gaa gaa gag 1675 Lys Lys Gly Ser GlyGlu Arg Arg Arg Val Tyr Arg Glu Glu Glu Glu 475 480 485 gaa gag gag ggccaa tac ccc cca gca cct cca cct tac tca gag act 1723 Glu Glu Glu Gly GlnTyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu Thr 490 495 500 gac tcg cag gcctca cgg gag agg agg ctg aaa aag aat ttg gcc ctg 1771 Asp Ser Gln Ala SerArg Glu Arg Arg Leu Lys Lys Asn Leu Ala Leu 505 510 515 agt cgg gaa agttta gtc gtc tga tccacgtttt gtatgtagct tttgtacttt 1825 Ser Arg Glu SerLeu Val Val * 520 525 ttttttaatt ggaatcaata ttgatgaaac ttcaagcctaataaaatgtc taatcacaaa 1885 aaaaaaaa 1893 6 525 PRT Rattus norvegicus 6Met Ala Pro Ala Ala Gly Ala Cys Ala Gly Ala Pro Asp Ser His Pro 1 5 1015 Ala Thr Val Val Phe Val Cys Leu Phe Leu Ile Ile Phe Cys Pro Asp 20 2530 Pro Ala Ser Ala Ile Gln Val Thr Val Ser Asp Pro Tyr His Val Val 35 4045 Ile Leu Phe Gln Pro Val Thr Leu Pro Cys Thr Tyr Gln Met Ser Asn 50 5560 Thr Leu Thr Val Pro Ile Val Ile Trp Lys Tyr Lys Ser Phe Cys Arg 65 7075 80 Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gln Leu 8590 95 Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val Glu100 105 110 Cys Gln Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys GlnGly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly Arg Arg IleThr Ile 130 135 140 Thr Gly Asn Ala Asp Leu Thr Phe Glu Gln Thr Ala TrpGly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Ser Ala GlnAsp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr Ala Glu Leu Ile Val LeuVal Tyr Ala Ala Gly Lys 180 185 190 Ala Ala Thr Ser Gly Val Pro Ser IleTyr Ala Pro Ser Ile Tyr Thr 195 200 205 His Leu Ser Pro Ala Lys Thr ProPro Pro Pro Pro Ala Met Ile Pro 210 215 220 Met Gly Pro Pro Tyr Gly TyrPro Gly Asp Phe Asp Arg His Ser Ser 225 230 235 240 Val Gly Gly His SerSer Gln Val Pro Leu Leu Arg Asp Val Asp Gly 245 250 255 Ser Val Ser SerGlu Val Arg Ser Gly Tyr Arg Ile Gln Ala Asn Gln 260 265 270 Gln Asp AspSer Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu Ala 275 280 285 Asn PheAsp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Val Glu Arg 290 295 300 AlaMet Ser Glu Val Thr Ser Leu His Glu Asp Asp Trp Arg Ser Arg 305 310 315320 Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp Glu Glu Trp Asn 325330 335 Arg His Ser Pro Gln Ser Pro Arg Thr Trp Glu Gln Glu Pro Leu Gln340 345 350 Glu Gln Pro Arg Gly Gly Trp Gly Ser Gly Arg Pro Arg Ala ArgSer 355 360 365 Val Asp Ala Leu Asp Asp Ile Asn Arg Pro Gly Ser Thr GluSer Gly 370 375 380 Arg Ser Ser Pro Pro Ser Ser Gly Arg Arg Gly Arg AlaTyr Ala Pro 385 390 395 400 Pro Arg Ser Arg Ser Arg Asp Asp Leu Tyr AspPro Asp Asp Pro Arg 405 410 415 Asp Leu Pro His Ser Arg Asp Pro His TyrTyr Asp Asp Ile Arg Ser 420 425 430 Arg Asp Pro Arg Ala Asp Pro Arg SerArg Gln Arg Ser Arg Asp Pro 435 440 445 Arg Asp Ala Gly Phe Arg Ser ArgAsp Pro Gln Tyr Asp Gly Arg Leu 450 455 460 Leu Glu Glu Ala Leu Lys LysLys Gly Ser Gly Glu Arg Arg Arg Val 465 470 475 480 Tyr Arg Glu Glu GluGlu Glu Glu Glu Gly Gln Tyr Pro Pro Ala Pro 485 490 495 Pro Pro Tyr SerGlu Thr Asp Ser Gln Ala Ser Arg Glu Arg Arg Leu 500 505 510 Lys Lys AsnLeu Ala Leu Ser Arg Glu Ser Leu Val Val 515 520 525 7 2158 DNA Homosapiens Misc_Feature 1259..1261 Potential splicing site AAG 7 tggagtgtggctcggaggac cgcggcgggt caagcacctt tctcccccat atctgaaagc 60 atgccctttgtccacgtcgt ttacgctcat taaaacttcc aga atg caa cag gac 115 Met Gln Gln Asp1 gga ctt gga gta ggg aca agg aac gga agt ggg aag ggg agg agc gtg 163Gly Leu Gly Val Gly Thr Arg Asn Gly Ser Gly Lys Gly Arg Ser Val 5 10 1520 cac ccc tcc tgg cct tgg tgc gcg ccg cgc ccc cta agg tac ttt gga 211His Pro Ser Trp Pro Trp Cys Ala Pro Arg Pro Leu Arg Tyr Phe Gly 25 30 35agg gac gcg cgg gcc aga cgc gcc cag acg gcc gcg atg gcg ctg ttg 259 ArgAsp Ala Arg Ala Arg Arg Ala Gln Thr Ala Ala Met Ala Leu Leu 40 45 50 gccggc ggg ctc tcc aga ggg ctg ggc tcc cac ccg gcc gcc gca ggc 307 Ala GlyGly Leu Ser Arg Gly Leu Gly Ser His Pro Ala Ala Ala Gly 55 60 65 cgg gacgcg gtc gtc ttc gtg tgg ctt ctg ctt agc acc tgg tgc aca 355 Arg Asp AlaVal Val Phe Val Trp Leu Leu Leu Ser Thr Trp Cys Thr 70 75 80 gct cct gccagg gcc atc cag gtg acc gtg tcc aac ccc tac cac gtg 403 Ala Pro Ala ArgAla Ile Gln Val Thr Val Ser Asn Pro Tyr His Val 85 90 95 100 gtg atc ctcttc cag cct gtg acc ctg ccc tgt acc tac cag atg acc 451 Val Ile Leu PheGln Pro Val Thr Leu Pro Cys Thr Tyr Gln Met Thr 105 110 115 tcg acc cccacg caa ccc atc gtc atc tgg aag tac aag tct ttc tgc 499 Ser Thr Pro ThrGln Pro Ile Val Ile Trp Lys Tyr Lys Ser Phe Cys 120 125 130 cgg gac cgcatc gcc gat gcc ttc tcc ccg gcc agc gtc gac aac cag 547 Arg Asp Arg IleAla Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gln 135 140 145 ctc aat gcccag ctg gca gcc ggg aac cca ggc tac aac ccc tac gtt 595 Leu Asn Ala GlnLeu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val 150 155 160 gag tgc caggac agc gtg cgc acc gtc agg gtc gtg gcc acc aag cag 643 Glu Cys Gln AspSer Val Arg Thr Val Arg Val Val Ala Thr Lys Gln 165 170 175 180 ggc aacgct gtg acc ctg gga gat tac tac cag ggc cgg agg att acc 691 Gly Asn AlaVal Thr Leu Gly Asp Tyr Tyr Gln Gly Arg Arg Ile Thr 185 190 195 atc accgga aat gct gac ctg acc ttt gac cag acg gcg tgg ggg gac 739 Ile Thr GlyAsn Ala Asp Leu Thr Phe Asp Gln Thr Ala Trp Gly Asp 200 205 210 agt ggtgtg tat tac tgc tcc gtg gtc tca gcc cag gac ctc cag ggg 787 Ser Gly ValTyr Tyr Cys Ser Val Val Ser Ala Gln Asp Leu Gln Gly 215 220 225 aac aatgag gcc tac gca gag ctc atc gtc ctt ggg agg acc tca ggg 835 Asn Asn GluAla Tyr Ala Glu Leu Ile Val Leu Gly Arg Thr Ser Gly 230 235 240 gtg gctgag ctc tta cct ggt ttt cag gcg ggg ccc ata gaa gac tgg 883 Val Ala GluLeu Leu Pro Gly Phe Gln Ala Gly Pro Ile Glu Asp Trp 245 250 255 260 ctcttc gtg gtt gtg gta tgc ctg gct gcc ttc ctc atc ttc ctc ctc 931 Leu PheVal Val Val Val Cys Leu Ala Ala Phe Leu Ile Phe Leu Leu 265 270 275 ctgggc atc tgc tgg tgc cag tgc tgc ccg cac act tgc tgc tgc tac 979 Leu GlyIle Cys Trp Cys Gln Cys Cys Pro His Thr Cys Cys Cys Tyr 280 285 290 gtcagg tgc ccc tgc tgc cca gac aag tgc tgc tgc ccc gag gcc ctg 1027 Val ArgCys Pro Cys Cys Pro Asp Lys Cys Cys Cys Pro Glu Ala Leu 295 300 305 tatgcc gcc ggc aaa gca gcc acc tca ggt gtt ccc agc att tat gcc 1075 Tyr AlaAla Gly Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala 310 315 320 cccagc acc tat gcc cac ctg tct ccc gcc aag acc cca ccc cca cca 1123 Pro SerThr Tyr Ala His Leu Ser Pro Ala Lys Thr Pro Pro Pro Pro 325 330 335 340gct atg att ccc atg ggc cct gcc tac aac ggg tac cct gga gga tac 1171 AlaMet Ile Pro Met Gly Pro Ala Tyr Asn Gly Tyr Pro Gly Gly Tyr 345 350 355cct gga gac gtt gac agg agt agc tca gct ggt ggc caa ggc tcc tat 1219 ProGly Asp Val Asp Arg Ser Ser Ser Ala Gly Gly Gln Gly Ser Tyr 360 365 370gta ccc ctg ctt cgg gac acg gac agc agt gtg gcc tct gaa gtc cgc 1267 ValPro Leu Leu Arg Asp Thr Asp Ser Ser Val Ala Ser Glu Val Arg 375 380 385agt ggc tac agg att cag gcc agc cag cag gac gac tcc atg cgg gtc 1315 SerGly Tyr Arg Ile Gln Ala Ser Gln Gln Asp Asp Ser Met Arg Val 390 395 400ctg tac tac atg gag aag gag ctg gcc aac ttc gac cct tct cga cct 1363 LeuTyr Tyr Met Glu Lys Glu Leu Ala Asn Phe Asp Pro Ser Arg Pro 405 410 415420 ggc ccc ccc agt ggc cgt gtg gag cgg gcc atg agt gaa gtc acc tcc 1411Gly Pro Pro Ser Gly Arg Val Glu Arg Ala Met Ser Glu Val Thr Ser 425 430435 ctc cac gag gac gac tgg cga tct cgg cct tcc cgg ggc cct gcc ctc 1459Leu His Glu Asp Asp Trp Arg Ser Arg Pro Ser Arg Gly Pro Ala Leu 440 445450 acc ccg atc cgg gat gag gag tgg ggt ggc cac tcc ccc cgg agt ccc 1507Thr Pro Ile Arg Asp Glu Glu Trp Gly Gly His Ser Pro Arg Ser Pro 455 460465 agg gga tgg gac cag gag ccc gcc agg gag cag gca ggc ggg ggc tgg 1555Arg Gly Trp Asp Gln Glu Pro Ala Arg Glu Gln Ala Gly Gly Gly Trp 470 475480 cgg gcc agg cgg ccc cgg gcc cgc tcc gtg gac gcc ctg gac gac ctc 1603Arg Ala Arg Arg Pro Arg Ala Arg Ser Val Asp Ala Leu Asp Asp Leu 485 490495 500 acc ccg ccg agc acc gcc gag tca ggg agc agg tct ccc acg agt aat1651 Thr Pro Pro Ser Thr Ala Glu Ser Gly Ser Arg Ser Pro Thr Ser Asn 505510 515 ggt ggg aga agc cgg gcc tac atg ccc ccg cgg agc cgc agc cgg gac1699 Gly Gly Arg Ser Arg Ala Tyr Met Pro Pro Arg Ser Arg Ser Arg Asp 520525 530 gac ctc tat gac caa gac gac tcg agg gac ttc cca cgc tcc cgg gac1747 Asp Leu Tyr Asp Gln Asp Asp Ser Arg Asp Phe Pro Arg Ser Arg Asp 535540 545 ccc cac tac gac gac ttc agg tct cgg gag cgc cct cct gcc gac ccc1795 Pro His Tyr Asp Asp Phe Arg Ser Arg Glu Arg Pro Pro Ala Asp Pro 550555 560 agg tcc cac cac cac cgt acc cgg gac cct cgg gac aac ggc tcc agg1843 Arg Ser His His His Arg Thr Arg Asp Pro Arg Asp Asn Gly Ser Arg 565570 575 580 tcc ggg gac ctc ccc tat gat ggg cgg cta ctg gag gag gct gtgagg 1891 Ser Gly Asp Leu Pro Tyr Asp Gly Arg Leu Leu Glu Glu Ala Val Arg585 590 595 aag aag ggg tcg gag gag agg agg aga ccc cac aag gag gag gaggaa 1939 Lys Lys Gly Ser Glu Glu Arg Arg Arg Pro His Lys Glu Glu Glu Glu600 605 610 gag gcc tac tac ccg ccc gcg ccg ccc ccg tac tcg gag acc gactcg 1987 Glu Ala Tyr Tyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu Thr Asp Ser615 620 625 cag gcg tcc cga gag cgc agg ctc aag aag aac ttg gcc ctg agtcgg 2035 Gln Ala Ser Arg Glu Arg Arg Leu Lys Lys Asn Leu Ala Leu Ser Arg630 635 640 gaa agt tta gtc gtc tga tctgacgttt tctacgtagc ttttgtattt2083 Glu Ser Leu Val Val * 645 ttttttttaa tttgaaggaa cactgatgaagccctgccat acccctcccg agtctaataa 2143 aacgtataat cacaa 2158 8 649 PRTHomo sapiens Misc_Feature 386 Potential deletion of a Glu 8 Met Gln GlnAsp Gly Leu Gly Val Gly Thr Arg Asn Gly Ser Gly Lys 1 5 10 15 Gly ArgSer Val His Pro Ser Trp Pro Trp Cys Ala Pro Arg Pro Leu 20 25 30 Arg TyrPhe Gly Arg Asp Ala Arg Ala Arg Arg Ala Gln Thr Ala Ala 35 40 45 Met AlaLeu Leu Ala Gly Gly Leu Ser Arg Gly Leu Gly Ser His Pro 50 55 60 Ala AlaAla Gly Arg Asp Ala Val Val Phe Val Trp Leu Leu Leu Ser 65 70 75 80 ThrTrp Cys Thr Ala Pro Ala Arg Ala Ile Gln Val Thr Val Ser Asn 85 90 95 ProTyr His Val Val Ile Leu Phe Gln Pro Val Thr Leu Pro Cys Thr 100 105 110Tyr Gln Met Thr Ser Thr Pro Thr Gln Pro Ile Val Ile Trp Lys Tyr 115 120125 Lys Ser Phe Cys Arg Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser 130135 140 Val Asp Asn Gln Leu Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly Tyr145 150 155 160 Asn Pro Tyr Val Glu Cys Gln Asp Ser Val Arg Thr Val ArgVal Val 165 170 175 Ala Thr Lys Gln Gly Asn Ala Val Thr Leu Gly Asp TyrTyr Gln Gly 180 185 190 Arg Arg Ile Thr Ile Thr Gly Asn Ala Asp Leu ThrPhe Asp Gln Thr 195 200 205 Ala Trp Gly Asp Ser Gly Val Tyr Tyr Cys SerVal Val Ser Ala Gln 210 215 220 Asp Leu Gln Gly Asn Asn Glu Ala Tyr AlaGlu Leu Ile Val Leu Gly 225 230 235 240 Arg Thr Ser Gly Val Ala Glu LeuLeu Pro Gly Phe Gln Ala Gly Pro 245 250 255 Ile Glu Asp Trp Leu Phe ValVal Val Val Cys Leu Ala Ala Phe Leu 260 265 270 Ile Phe Leu Leu Leu GlyIle Cys Trp Cys Gln Cys Cys Pro His Thr 275 280 285 Cys Cys Cys Tyr ValArg Cys Pro Cys Cys Pro Asp Lys Cys Cys Cys 290 295 300 Pro Glu Ala LeuTyr Ala Ala Gly Lys Ala Ala Thr Ser Gly Val Pro 305 310 315 320 Ser IleTyr Ala Pro Ser Thr Tyr Ala His Leu Ser Pro Ala Lys Thr 325 330 335 ProPro Pro Pro Ala Met Ile Pro Met Gly Pro Ala Tyr Asn Gly Tyr 340 345 350Pro Gly Gly Tyr Pro Gly Asp Val Asp Arg Ser Ser Ser Ala Gly Gly 355 360365 Gln Gly Ser Tyr Val Pro Leu Leu Arg Asp Thr Asp Ser Ser Val Ala 370375 380 Ser Glu Val Arg Ser Gly Tyr Arg Ile Gln Ala Ser Gln Gln Asp Asp385 390 395 400 Ser Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu Ala AsnPhe Asp 405 410 415 Pro Ser Arg Pro Gly Pro Pro Ser Gly Arg Val Glu ArgAla Met Ser 420 425 430 Glu Val Thr Ser Leu His Glu Asp Asp Trp Arg SerArg Pro Ser Arg 435 440 445 Gly Pro Ala Leu Thr Pro Ile Arg Asp Glu GluTrp Gly Gly His Ser 450 455 460 Pro Arg Ser Pro Arg Gly Trp Asp Gln GluPro Ala Arg Glu Gln Ala 465 470 475 480 Gly Gly Gly Trp Arg Ala Arg ArgPro Arg Ala Arg Ser Val Asp Ala 485 490 495 Leu Asp Asp Leu Thr Pro ProSer Thr Ala Glu Ser Gly Ser Arg Ser 500 505 510 Pro Thr Ser Asn Gly GlyArg Ser Arg Ala Tyr Met Pro Pro Arg Ser 515 520 525 Arg Ser Arg Asp AspLeu Tyr Asp Gln Asp Asp Ser Arg Asp Phe Pro 530 535 540 Arg Ser Arg AspPro His Tyr Asp Asp Phe Arg Ser Arg Glu Arg Pro 545 550 555 560 Pro AlaAsp Pro Arg Ser His His His Arg Thr Arg Asp Pro Arg Asp 565 570 575 AsnGly Ser Arg Ser Gly Asp Leu Pro Tyr Asp Gly Arg Leu Leu Glu 580 585 590Glu Ala Val Arg Lys Lys Gly Ser Glu Glu Arg Arg Arg Pro His Lys 595 600605 Glu Glu Glu Glu Glu Ala Tyr Tyr Pro Pro Ala Pro Pro Pro Tyr Ser 610615 620 Glu Thr Asp Ser Gln Ala Ser Arg Glu Arg Arg Leu Lys Lys Asn Leu625 630 635 640 Ala Leu Ser Arg Glu Ser Leu Val Val 645 9 2101 DNA Homosapiens 9 tggagtgtgg ctcggaggac cgcggcgggt caagcacctt tctcccccatatctgaaagc 60 atgccctttg tccacgtcgt ttacgctcat taaaacttcc aga atg caacag gac 115 Met Gln Gln Asp 1 gga ctt gga gta ggg aca agg aac gga agtggg aag ggg agg agc gtg 163 Gly Leu Gly Val Gly Thr Arg Asn Gly Ser GlyLys Gly Arg Ser Val 5 10 15 20 cac ccc tcc tgg cct tgg tgc gcg ccg cgcccc cta agg tac ttt gga 211 His Pro Ser Trp Pro Trp Cys Ala Pro Arg ProLeu Arg Tyr Phe Gly 25 30 35 agg gac gcg cgg gcc aga cgc gcc cag acg gccgcg atg gcg ctg ttg 259 Arg Asp Ala Arg Ala Arg Arg Ala Gln Thr Ala AlaMet Ala Leu Leu 40 45 50 gcc ggc ggg ctc tcc aga ggg ctg ggc tcc cac ccggcc gcc gca ggc 307 Ala Gly Gly Leu Ser Arg Gly Leu Gly Ser His Pro AlaAla Ala Gly 55 60 65 cgg gac gcg gtc gtc ttc gtg tgg ctt ctg ctt agc acctgg tgc aca 355 Arg Asp Ala Val Val Phe Val Trp Leu Leu Leu Ser Thr TrpCys Thr 70 75 80 gct cct gcc agg gcc atc cag gtg acc gtg tcc aac ccc taccac gtg 403 Ala Pro Ala Arg Ala Ile Gln Val Thr Val Ser Asn Pro Tyr HisVal 85 90 95 100 gtg atc ctc ttc cag cct gtg acc ctg ccc tgt acc tac cagatg acc 451 Val Ile Leu Phe Gln Pro Val Thr Leu Pro Cys Thr Tyr Gln MetThr 105 110 115 tcg acc ccc acg caa ccc atc gtc atc tgg aag tac aag tctttc tgc 499 Ser Thr Pro Thr Gln Pro Ile Val Ile Trp Lys Tyr Lys Ser PheCys 120 125 130 cgg gac cgc atc gcc gat gcc ttc tcc ccg gcc agc gtc gacaac cag 547 Arg Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser Val Asp AsnGln 135 140 145 ctc aat gcc cag ctg gca gcc ggg aac cca ggc tac aac ccctac gtt 595 Leu Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro TyrVal 150 155 160 gag tgc cag gac agc gtg cgc acc gtc agg gtc gtg gcc accaag cag 643 Glu Cys Gln Asp Ser Val Arg Thr Val Arg Val Val Ala Thr LysGln 165 170 175 180 ggc aac gct gtg acc ctg gga gat tac tac cag ggc cggagg att acc 691 Gly Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly Arg ArgIle Thr 185 190 195 atc acc gga aat gct gac ctg acc ttt gac cag acg gcgtgg ggg gac 739 Ile Thr Gly Asn Ala Asp Leu Thr Phe Asp Gln Thr Ala TrpGly Asp 200 205 210 agt ggt gtg tat tac tgc tcc gtg gtc tca gcc cag gacctc cag ggg 787 Ser Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gln Asp LeuGln Gly 215 220 225 aac aat gag gcc tac gca gag ctc atc gtc ctt gac tggctc ttc gtg 835 Asn Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Asp Trp LeuPhe Val 230 235 240 gtt gtg gta tgc ctg gct gcc ttc ctc atc ttc ctc ctcctg ggc atc 883 Val Val Val Cys Leu Ala Ala Phe Leu Ile Phe Leu Leu LeuGly Ile 245 250 255 260 tgc tgg tgc cag tgc tgc ccg cac act tgc tgc tgctac gtc agg tgc 931 Cys Trp Cys Gln Cys Cys Pro His Thr Cys Cys Cys TyrVal Arg Cys 265 270 275 ccc tgc tgc cca gac aag tgc tgc tgc ccc gag gccctg tat gcc gcc 979 Pro Cys Cys Pro Asp Lys Cys Cys Cys Pro Glu Ala LeuTyr Ala Ala 280 285 290 ggc aaa gca gcc acc tca ggt gtt ccc agc att tatgcc ccc agc acc 1027 Gly Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr AlaPro Ser Thr 295 300 305 tat gcc cac ctg tct ccc gcc aag acc cca ccc ccacca gct atg att 1075 Tyr Ala His Leu Ser Pro Ala Lys Thr Pro Pro Pro ProAla Met Ile 310 315 320 ccc atg ggc cct gcc tac aac ggg tac cct gga ggatac cct gga gac 1123 Pro Met Gly Pro Ala Tyr Asn Gly Tyr Pro Gly Gly TyrPro Gly Asp 325 330 335 340 gtt gac agg agt agc tca gct ggt ggc caa ggctcc tat gta ccc ctg 1171 Val Asp Arg Ser Ser Ser Ala Gly Gly Gln Gly SerTyr Val Pro Leu 345 350 355 ctt cgg gac acg gac agc agt gtg gcc tct gaagtc cgc agt ggc tac 1219 Leu Arg Asp Thr Asp Ser Ser Val Ala Ser Glu ValArg Ser Gly Tyr 360 365 370 agg att cag gcc agc cag cag gac gac tcc atgcgg gtc ctg tac tac 1267 Arg Ile Gln Ala Ser Gln Gln Asp Asp Ser Met ArgVal Leu Tyr Tyr 375 380 385 atg gag aag gag ctg gcc aac ttc gac cct tctcga cct ggc ccc ccc 1315 Met Glu Lys Glu Leu Ala Asn Phe Asp Pro Ser ArgPro Gly Pro Pro 390 395 400 agt ggc cgt gtg gag cgg gcc atg agt gaa gtcacc tcc ctc cac gag 1363 Ser Gly Arg Val Glu Arg Ala Met Ser Glu Val ThrSer Leu His Glu 405 410 415 420 gac gac tgg cga tct cgg cct tcc cgg ggccct gcc ctc acc ccg atc 1411 Asp Asp Trp Arg Ser Arg Pro Ser Arg Gly ProAla Leu Thr Pro Ile 425 430 435 cgg gat gag gag tgg ggt ggc cac tcc ccccgg agt ccc agg gga tgg 1459 Arg Asp Glu Glu Trp Gly Gly His Ser Pro ArgSer Pro Arg Gly Trp 440 445 450 gac cag gag ccc gcc agg gag cag gca ggcggg ggc tgg cgg gcc agg 1507 Asp Gln Glu Pro Ala Arg Glu Gln Ala Gly GlyGly Trp Arg Ala Arg 455 460 465 cgg ccc cgg gcc cgc tcc gtg gac gcc ctggac gac ctc acc ccg ccg 1555 Arg Pro Arg Ala Arg Ser Val Asp Ala Leu AspAsp Leu Thr Pro Pro 470 475 480 agc acc gcc gag tca ggg agc agg tct cccacg agt aat ggt ggg aga 1603 Ser Thr Ala Glu Ser Gly Ser Arg Ser Pro ThrSer Asn Gly Gly Arg 485 490 495 500 agc cgg gcc tac atg ccc ccg cgg agccgc agc cgg gac gac ctc tat 1651 Ser Arg Ala Tyr Met Pro Pro Arg Ser ArgSer Arg Asp Asp Leu Tyr 505 510 515 gac caa gac gac tcg agg gac ttc ccacgc tcc cgg gac ccc cac tac 1699 Asp Gln Asp Asp Ser Arg Asp Phe Pro ArgSer Arg Asp Pro His Tyr 520 525 530 gac gac ttc agg tct cgg gag cgc cctcct gcc gac ccc agg tcc cac 1747 Asp Asp Phe Arg Ser Arg Glu Arg Pro ProAla Asp Pro Arg Ser His 535 540 545 cac cac cgt acc cgg gac cct cgg gacaac ggc tcc agg tcc ggg gac 1795 His His Arg Thr Arg Asp Pro Arg Asp AsnGly Ser Arg Ser Gly Asp 550 555 560 ctc ccc tat gat ggg cgg cta ctg gaggag gct gtg agg aag aag ggg 1843 Leu Pro Tyr Asp Gly Arg Leu Leu Glu GluAla Val Arg Lys Lys Gly 565 570 575 580 tcg gag gag agg agg aga ccc cacaag gag gag gag gaa gag gcc tac 1891 Ser Glu Glu Arg Arg Arg Pro His LysGlu Glu Glu Glu Glu Ala Tyr 585 590 595 tac ccg ccc gcg ccg ccc ccg tactcg gag acc gac tcg cag gcg tcc 1939 Tyr Pro Pro Ala Pro Pro Pro Tyr SerGlu Thr Asp Ser Gln Ala Ser 600 605 610 cga gag cgc agg ctc aag aag aacttg gcc ctg agt cgg gaa agt tta 1987 Arg Glu Arg Arg Leu Lys Lys Asn LeuAla Leu Ser Arg Glu Ser Leu 615 620 625 gtc gtc tga tctgacgttttctacgtagc ttttgtattt ttttttttaa 2036 Val Val * 630 tttgaaggaacactgatgaa gccctgccat acccctcccg agtctaataa aacgtataat 2096 cacaa 210110 630 PRT Homo sapiens 10 Met Gln Gln Asp Gly Leu Gly Val Gly Thr ArgAsn Gly Ser Gly Lys 1 5 10 15 Gly Arg Ser Val His Pro Ser Trp Pro TrpCys Ala Pro Arg Pro Leu 20 25 30 Arg Tyr Phe Gly Arg Asp Ala Arg Ala ArgArg Ala Gln Thr Ala Ala 35 40 45 Met Ala Leu Leu Ala Gly Gly Leu Ser ArgGly Leu Gly Ser His Pro 50 55 60 Ala Ala Ala Gly Arg Asp Ala Val Val PheVal Trp Leu Leu Leu Ser 65 70 75 80 Thr Trp Cys Thr Ala Pro Ala Arg AlaIle Gln Val Thr Val Ser Asn 85 90 95 Pro Tyr His Val Val Ile Leu Phe GlnPro Val Thr Leu Pro Cys Thr 100 105 110 Tyr Gln Met Thr Ser Thr Pro ThrGln Pro Ile Val Ile Trp Lys Tyr 115 120 125 Lys Ser Phe Cys Arg Asp ArgIle Ala Asp Ala Phe Ser Pro Ala Ser 130 135 140 Val Asp Asn Gln Leu AsnAla Gln Leu Ala Ala Gly Asn Pro Gly Tyr 145 150 155 160 Asn Pro Tyr ValGlu Cys Gln Asp Ser Val Arg Thr Val Arg Val Val 165 170 175 Ala Thr LysGln Gly Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly 180 185 190 Arg ArgIle Thr Ile Thr Gly Asn Ala Asp Leu Thr Phe Asp Gln Thr 195 200 205 AlaTrp Gly Asp Ser Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gln 210 215 220Asp Leu Gln Gly Asn Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Asp 225 230235 240 Trp Leu Phe Val Val Val Val Cys Leu Ala Ala Phe Leu Ile Phe Leu245 250 255 Leu Leu Gly Ile Cys Trp Cys Gln Cys Cys Pro His Thr Cys CysCys 260 265 270 Tyr Val Arg Cys Pro Cys Cys Pro Asp Lys Cys Cys Cys ProGlu Ala 275 280 285 Leu Tyr Ala Ala Gly Lys Ala Ala Thr Ser Gly Val ProSer Ile Tyr 290 295 300 Ala Pro Ser Thr Tyr Ala His Leu Ser Pro Ala LysThr Pro Pro Pro 305 310 315 320 Pro Ala Met Ile Pro Met Gly Pro Ala TyrAsn Gly Tyr Pro Gly Gly 325 330 335 Tyr Pro Gly Asp Val Asp Arg Ser SerSer Ala Gly Gly Gln Gly Ser 340 345 350 Tyr Val Pro Leu Leu Arg Asp ThrAsp Ser Ser Val Ala Ser Glu Val 355 360 365 Arg Ser Gly Tyr Arg Ile GlnAla Ser Gln Gln Asp Asp Ser Met Arg 370 375 380 Val Leu Tyr Tyr Met GluLys Glu Leu Ala Asn Phe Asp Pro Ser Arg 385 390 395 400 Pro Gly Pro ProSer Gly Arg Val Glu Arg Ala Met Ser Glu Val Thr 405 410 415 Ser Leu HisGlu Asp Asp Trp Arg Ser Arg Pro Ser Arg Gly Pro Ala 420 425 430 Leu ThrPro Ile Arg Asp Glu Glu Trp Gly Gly His Ser Pro Arg Ser 435 440 445 ProArg Gly Trp Asp Gln Glu Pro Ala Arg Glu Gln Ala Gly Gly Gly 450 455 460Trp Arg Ala Arg Arg Pro Arg Ala Arg Ser Val Asp Ala Leu Asp Asp 465 470475 480 Leu Thr Pro Pro Ser Thr Ala Glu Ser Gly Ser Arg Ser Pro Thr Ser485 490 495 Asn Gly Gly Arg Ser Arg Ala Tyr Met Pro Pro Arg Ser Arg SerArg 500 505 510 Asp Asp Leu Tyr Asp Gln Asp Asp Ser Arg Asp Phe Pro ArgSer Arg 515 520 525 Asp Pro His Tyr Asp Asp Phe Arg Ser Arg Glu Arg ProPro Ala Asp 530 535 540 Pro Arg Ser His His His Arg Thr Arg Asp Pro ArgAsp Asn Gly Ser 545 550 555 560 Arg Ser Gly Asp Leu Pro Tyr Asp Gly ArgLeu Leu Glu Glu Ala Val 565 570 575 Arg Lys Lys Gly Ser Glu Glu Arg ArgArg Pro His Lys Glu Glu Glu 580 585 590 Glu Glu Ala Tyr Tyr Pro Pro AlaPro Pro Pro Tyr Ser Glu Thr Asp 595 600 605 Ser Gln Ala Ser Arg Glu ArgArg Leu Lys Lys Asn Leu Ala Leu Ser 610 615 620 Arg Glu Ser Leu Val Val625 630 11 1954 DNA Homo sapiens 11 tggagtgtgg ctcggaggac cgcggcgggtcaagcacctt tctcccccat atctgaaagc 60 atgccctttg tccacgtcgt ttacgctcattaaaacttcc aga atg caa cag gac 115 Met Gln Gln Asp 1 gga ctt gga gta gggaca agg aac gga agt ggg aag ggg agg agc gtg 163 Gly Leu Gly Val Gly ThrArg Asn Gly Ser Gly Lys Gly Arg Ser Val 5 10 15 20 cac ccc tcc tgg ccttgg tgc gcg ccg cgc ccc cta agg tac ttt gga 211 His Pro Ser Trp Pro TrpCys Ala Pro Arg Pro Leu Arg Tyr Phe Gly 25 30 35 agg gac gcg cgg gcc agacgc gcc cag acg gcc gcg atg gcg ctg ttg 259 Arg Asp Ala Arg Ala Arg ArgAla Gln Thr Ala Ala Met Ala Leu Leu 40 45 50 gcc ggc ggg ctc tcc aga gggctg ggc tcc cac ccg gcc gcc gca ggc 307 Ala Gly Gly Leu Ser Arg Gly LeuGly Ser His Pro Ala Ala Ala Gly 55 60 65 cgg gac gcg gtc gtc ttc gtg tggctt ctg ctt agc acc tgg tgc aca 355 Arg Asp Ala Val Val Phe Val Trp LeuLeu Leu Ser Thr Trp Cys Thr 70 75 80 gct cct gcc agg gcc atc cag gtg accgtg tcc aac ccc tac cac gtg 403 Ala Pro Ala Arg Ala Ile Gln Val Thr ValSer Asn Pro Tyr His Val 85 90 95 100 gtg atc ctc ttc cag cct gtg acc ctgccc tgt acc tac cag atg acc 451 Val Ile Leu Phe Gln Pro Val Thr Leu ProCys Thr Tyr Gln Met Thr 105 110 115 tcg acc ccc acg caa ccc atc gtc atctgg aag tac aag tct ttc tgc 499 Ser Thr Pro Thr Gln Pro Ile Val Ile TrpLys Tyr Lys Ser Phe Cys 120 125 130 cgg gac cgc atc gcc gat gcc ttc tccccg gcc agc gtc gac aac cag 547 Arg Asp Arg Ile Ala Asp Ala Phe Ser ProAla Ser Val Asp Asn Gln 135 140 145 ctc aat gcc cag ctg gca gcc ggg aaccca ggc tac aac ccc tac gtt 595 Leu Asn Ala Gln Leu Ala Ala Gly Asn ProGly Tyr Asn Pro Tyr Val 150 155 160 gag tgc cag gac agc gtg cgc acc gtcagg gtc gtg gcc acc aag cag 643 Glu Cys Gln Asp Ser Val Arg Thr Val ArgVal Val Ala Thr Lys Gln 165 170 175 180 ggc aac gct gtg acc ctg gga gattac tac cag ggc cgg agg att acc 691 Gly Asn Ala Val Thr Leu Gly Asp TyrTyr Gln Gly Arg Arg Ile Thr 185 190 195 atc acc gga aat gct gac ctg accttt gac cag acg gcg tgg ggg gac 739 Ile Thr Gly Asn Ala Asp Leu Thr PheAsp Gln Thr Ala Trp Gly Asp 200 205 210 agt ggt gtg tat tac tgc tcc gtggtc tca gcc cag gac ctc cag ggg 787 Ser Gly Val Tyr Tyr Cys Ser Val ValSer Ala Gln Asp Leu Gln Gly 215 220 225 aac aat gag gcc tac gca gag ctcatc gtc ctt gtg tat gcc gcc ggc 835 Asn Asn Glu Ala Tyr Ala Glu Leu IleVal Leu Val Tyr Ala Ala Gly 230 235 240 aaa gca gcc acc tca ggt gtt cccagc att tat gcc ccc agc acc tat 883 Lys Ala Ala Thr Ser Gly Val Pro SerIle Tyr Ala Pro Ser Thr Tyr 245 250 255 260 gcc cac ctg tct ccc gcc aagacc cca ccc cca cca gct atg att ccc 931 Ala His Leu Ser Pro Ala Lys ThrPro Pro Pro Pro Ala Met Ile Pro 265 270 275 atg ggc cct gcc tac aac gggtac cct gga gga tac cct gga gac gtt 979 Met Gly Pro Ala Tyr Asn Gly TyrPro Gly Gly Tyr Pro Gly Asp Val 280 285 290 gac agg agt agc tca gct ggtggc caa ggc tcc tat gta ccc ctg ctt 1027 Asp Arg Ser Ser Ser Ala Gly GlyGln Gly Ser Tyr Val Pro Leu Leu 295 300 305 cgg gac acg gac agc agt gtggcc tct gaa gtc cgc agt ggc tac agg 1075 Arg Asp Thr Asp Ser Ser Val AlaSer Glu Val Arg Ser Gly Tyr Arg 310 315 320 att cag gcc agc cag cag gacgac tcc atg cgg gtc ctg tac tac atg 1123 Ile Gln Ala Ser Gln Gln Asp AspSer Met Arg Val Leu Tyr Tyr Met 325 330 335 340 gag aag gag ctg gcc aacttc gac cct tct cga cct ggc ccc ccc agt 1171 Glu Lys Glu Leu Ala Asn PheAsp Pro Ser Arg Pro Gly Pro Pro Ser 345 350 355 ggc cgt gtg gag cgg gccatg agt gaa gtc acc tcc ctc cac gag gac 1219 Gly Arg Val Glu Arg Ala MetSer Glu Val Thr Ser Leu His Glu Asp 360 365 370 gac tgg cga tct cgg ccttcc cgg ggc cct gcc ctc acc ccg atc cgg 1267 Asp Trp Arg Ser Arg Pro SerArg Gly Pro Ala Leu Thr Pro Ile Arg 375 380 385 gat gag gag tgg ggt ggccac tcc ccc cgg agt ccc agg gga tgg gac 1315 Asp Glu Glu Trp Gly Gly HisSer Pro Arg Ser Pro Arg Gly Trp Asp 390 395 400 cag gag ccc gcc agg gagcag gca ggc ggg ggc tgg cgg gcc agg cgg 1363 Gln Glu Pro Ala Arg Glu GlnAla Gly Gly Gly Trp Arg Ala Arg Arg 405 410 415 420 ccc cgg gcc cgc tccgtg gac gcc ctg gac gac ctc acc ccg ccg agc 1411 Pro Arg Ala Arg Ser ValAsp Ala Leu Asp Asp Leu Thr Pro Pro Ser 425 430 435 acc gcc gag tca gggagc agg tct ccc acg agt aat ggt ggg aga agc 1459 Thr Ala Glu Ser Gly SerArg Ser Pro Thr Ser Asn Gly Gly Arg Ser 440 445 450 cgg gcc tac atg cccccg cgg agc cgc agc cgg gac gac ctc tat gac 1507 Arg Ala Tyr Met Pro ProArg Ser Arg Ser Arg Asp Asp Leu Tyr Asp 455 460 465 caa gac gac tcg agggac ttc cca cgc tcc cgg gac ccc cac tac gac 1555 Gln Asp Asp Ser Arg AspPhe Pro Arg Ser Arg Asp Pro His Tyr Asp 470 475 480 gac ttc agg tct cgggag cgc cct cct gcc gac ccc agg tcc cac cac 1603 Asp Phe Arg Ser Arg GluArg Pro Pro Ala Asp Pro Arg Ser His His 485 490 495 500 cac cgt acc cgggac cct cgg gac aac ggc tcc agg tcc ggg gac ctc 1651 His Arg Thr Arg AspPro Arg Asp Asn Gly Ser Arg Ser Gly Asp Leu 505 510 515 ccc tat gat gggcgg cta ctg gag gag gct gtg agg aag aag ggg tcg 1699 Pro Tyr Asp Gly ArgLeu Leu Glu Glu Ala Val Arg Lys Lys Gly Ser 520 525 530 gag gag agg aggaga ccc cac aag gag gag gag gaa gag gcc tac tac 1747 Glu Glu Arg Arg ArgPro His Lys Glu Glu Glu Glu Glu Ala Tyr Tyr 535 540 545 ccg ccc gcg ccgccc ccg tac tcg gag acc gac tcg cag gcg tcc cga 1795 Pro Pro Ala Pro ProPro Tyr Ser Glu Thr Asp Ser Gln Ala Ser Arg 550 555 560 gag cgc agg ctcaag aag aac ttg gcc ctg agt cgg gaa agt tta gtc 1843 Glu Arg Arg Leu LysLys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val 565 570 575 580 gtc tgatctgacgttt tctacgtagc ttttgtattt ttttttttaa tttgaaggaa 1899 Val *cactgatgaa gccctgccat acccctcccg agtctaataa aacgtataat cacaa 1954 12 581PRT Homo sapiens 12 Met Gln Gln Asp Gly Leu Gly Val Gly Thr Arg Asn GlySer Gly Lys 1 5 10 15 Gly Arg Ser Val His Pro Ser Trp Pro Trp Cys AlaPro Arg Pro Leu 20 25 30 Arg Tyr Phe Gly Arg Asp Ala Arg Ala Arg Arg AlaGln Thr Ala Ala 35 40 45 Met Ala Leu Leu Ala Gly Gly Leu Ser Arg Gly LeuGly Ser His Pro 50 55 60 Ala Ala Ala Gly Arg Asp Ala Val Val Phe Val TrpLeu Leu Leu Ser 65 70 75 80 Thr Trp Cys Thr Ala Pro Ala Arg Ala Ile GlnVal Thr Val Ser Asn 85 90 95 Pro Tyr His Val Val Ile Leu Phe Gln Pro ValThr Leu Pro Cys Thr 100 105 110 Tyr Gln Met Thr Ser Thr Pro Thr Gln ProIle Val Ile Trp Lys Tyr 115 120 125 Lys Ser Phe Cys Arg Asp Arg Ile AlaAsp Ala Phe Ser Pro Ala Ser 130 135 140 Val Asp Asn Gln Leu Asn Ala GlnLeu Ala Ala Gly Asn Pro Gly Tyr 145 150 155 160 Asn Pro Tyr Val Glu CysGln Asp Ser Val Arg Thr Val Arg Val Val 165 170 175 Ala Thr Lys Gln GlyAsn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly 180 185 190 Arg Arg Ile ThrIle Thr Gly Asn Ala Asp Leu Thr Phe Asp Gln Thr 195 200 205 Ala Trp GlyAsp Ser Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gln 210 215 220 Asp LeuGln Gly Asn Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Val 225 230 235 240Tyr Ala Ala Gly Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala 245 250255 Pro Ser Thr Tyr Ala His Leu Ser Pro Ala Lys Thr Pro Pro Pro Pro 260265 270 Ala Met Ile Pro Met Gly Pro Ala Tyr Asn Gly Tyr Pro Gly Gly Tyr275 280 285 Pro Gly Asp Val Asp Arg Ser Ser Ser Ala Gly Gly Gln Gly SerTyr 290 295 300 Val Pro Leu Leu Arg Asp Thr Asp Ser Ser Val Ala Ser GluVal Arg 305 310 315 320 Ser Gly Tyr Arg Ile Gln Ala Ser Gln Gln Asp AspSer Met Arg Val 325 330 335 Leu Tyr Tyr Met Glu Lys Glu Leu Ala Asn PheAsp Pro Ser Arg Pro 340 345 350 Gly Pro Pro Ser Gly Arg Val Glu Arg AlaMet Ser Glu Val Thr Ser 355 360 365 Leu His Glu Asp Asp Trp Arg Ser ArgPro Ser Arg Gly Pro Ala Leu 370 375 380 Thr Pro Ile Arg Asp Glu Glu TrpGly Gly His Ser Pro Arg Ser Pro 385 390 395 400 Arg Gly Trp Asp Gln GluPro Ala Arg Glu Gln Ala Gly Gly Gly Trp 405 410 415 Arg Ala Arg Arg ProArg Ala Arg Ser Val Asp Ala Leu Asp Asp Leu 420 425 430 Thr Pro Pro SerThr Ala Glu Ser Gly Ser Arg Ser Pro Thr Ser Asn 435 440 445 Gly Gly ArgSer Arg Ala Tyr Met Pro Pro Arg Ser Arg Ser Arg Asp 450 455 460 Asp LeuTyr Asp Gln Asp Asp Ser Arg Asp Phe Pro Arg Ser Arg Asp 465 470 475 480Pro His Tyr Asp Asp Phe Arg Ser Arg Glu Arg Pro Pro Ala Asp Pro 485 490495 Arg Ser His His His Arg Thr Arg Asp Pro Arg Asp Asn Gly Ser Arg 500505 510 Ser Gly Asp Leu Pro Tyr Asp Gly Arg Leu Leu Glu Glu Ala Val Arg515 520 525 Lys Lys Gly Ser Glu Glu Arg Arg Arg Pro His Lys Glu Glu GluGlu 530 535 540 Glu Ala Tyr Tyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu ThrAsp Ser 545 550 555 560 Gln Ala Ser Arg Glu Arg Arg Leu Lys Lys Asn LeuAla Leu Ser Arg 565 570 575 Glu Ser Leu Val Val 580 13 1886 DNA Musmusculus 13 gcaccgtcgc tgctagacgg ccgcg atg gcg ccg gcg gcc agc gcg tgtgct 52 Met Ala Pro Ala Ala Ser Ala Cys Ala 1 5 ggg gcg cct ggc tcc cacccg gcc acc acg atc ttc gtg tgt ctt ttt 100 Gly Ala Pro Gly Ser His ProAla Thr Thr Ile Phe Val Cys Leu Phe 10 15 20 25 ctc atc att tac tgc ccagac cgt gcc agt gcc atc cag gtg acc gtg 148 Leu Ile Ile Tyr Cys Pro AspArg Ala Ser Ala Ile Gln Val Thr Val 30 35 40 cct gac ccc tac cac gta gtgatc ctg ttc cag cca gtg aca cta cac 196 Pro Asp Pro Tyr His Val Val IleLeu Phe Gln Pro Val Thr Leu His 45 50 55 tgc acc tac cag atg agc aat accctc aca gcc cct atc gtg atc tgg 244 Cys Thr Tyr Gln Met Ser Asn Thr LeuThr Ala Pro Ile Val Ile Trp 60 65 70 aag tat aag tcg ttc tgt cgg gac cgtgtt gcc gac gcc ttc tcc cct 292 Lys Tyr Lys Ser Phe Cys Arg Asp Arg ValAla Asp Ala Phe Ser Pro 75 80 85 gcc agc gtg gac aac cag ctc aac gcc cagctg gcg gct ggc aac ccc 340 Ala Ser Val Asp Asn Gln Leu Asn Ala Gln LeuAla Ala Gly Asn Pro 90 95 100 105 ggc tac aac ccc tat gtg gag tgc caggac agc gta cgc act gtc agg 388 Gly Tyr Asn Pro Tyr Val Glu Cys Gln AspSer Val Arg Thr Val Arg 110 115 120 gtg gtg gcc acc aaa cag ggc aat gctgtg acc ctg gga gac tac tac 436 Val Val Ala Thr Lys Gln Gly Asn Ala ValThr Leu Gly Asp Tyr Tyr 125 130 135 cag ggc agg aga atc acc atc aca ggaaat gct ggc ctg acc ttc gag 484 Gln Gly Arg Arg Ile Thr Ile Thr Gly AsnAla Gly Leu Thr Phe Glu 140 145 150 cag acg gcc tgg gga gac agt gga gtgtat tac tgc tcc gtg gtc tca 532 Gln Thr Ala Trp Gly Asp Ser Gly Val TyrTyr Cys Ser Val Val Ser 155 160 165 gcc caa gat ctg gat ggg aac aac gaggcg tac gca gag ctc att gtc 580 Ala Gln Asp Leu Asp Gly Asn Asn Glu AlaTyr Ala Glu Leu Ile Val 170 175 180 185 ctt ggc agg acc tca gaa gcc cctgag ctc cta cct ggt ttt cgg gcg 628 Leu Gly Arg Thr Ser Glu Ala Pro GluLeu Leu Pro Gly Phe Arg Ala 190 195 200 ggg ccc ttg gaa gat tgg ctc tttgtg gtc gtg gtc tgc ctg gca agc 676 Gly Pro Leu Glu Asp Trp Leu Phe ValVal Val Val Cys Leu Ala Ser 205 210 215 ctc ctc ttc ttc ctc ctc ctg ggcatc tgc tgg tgc cag tgc tgt ccc 724 Leu Leu Phe Phe Leu Leu Leu Gly IleCys Trp Cys Gln Cys Cys Pro 220 225 230 cac acc tgc tgc tgc tat gtc agatgt ccc tgc tgc cca gac aag tgc 772 His Thr Cys Cys Cys Tyr Val Arg CysPro Cys Cys Pro Asp Lys Cys 235 240 245 tgt tgc cct gag gcc ctt tat gctgct ggc aaa gca gcc acc tca ggt 820 Cys Cys Pro Glu Ala Leu Tyr Ala AlaGly Lys Ala Ala Thr Ser Gly 250 255 260 265 gtg cca agc atc tat gcc cccagc atc tat acc cac ctc tct cct gcc 868 Val Pro Ser Ile Tyr Ala Pro SerIle Tyr Thr His Leu Ser Pro Ala 270 275 280 aag act ccg cca cct ccg cctgcc atg att ccc atg cgt cct ccc tat 916 Lys Thr Pro Pro Pro Pro Pro AlaMet Ile Pro Met Arg Pro Pro Tyr 285 290 295 ggg tac cct gga gac ttt gacagg acc agc tca gtt ggt ggc cac agc 964 Gly Tyr Pro Gly Asp Phe Asp ArgThr Ser Ser Val Gly Gly His Ser 300 305 310 tcc cag gtg ccc ctg ctg cgtgaa gtg gat ggg agc gta tct tca gaa 1012 Ser Gln Val Pro Leu Leu Arg GluVal Asp Gly Ser Val Ser Ser Glu 315 320 325 gta cga agt ggc tac agg atccag gct aac cag caa gat gac tcc atg 1060 Val Arg Ser Gly Tyr Arg Ile GlnAla Asn Gln Gln Asp Asp Ser Met 330 335 340 345 agg gtc cta tac tat atggag aag gag cta gcc aac ttc gat cct tcc 1108 Arg Val Leu Tyr Tyr Met GluLys Glu Leu Ala Asn Phe Asp Pro Ser 350 355 360 cgg cct ggc cct ccc aatggc cga gtg gaa cgg gcc atg agt gaa gta 1156 Arg Pro Gly Pro Pro Asn GlyArg Val Glu Arg Ala Met Ser Glu Val 365 370 375 acc tcc ctc cat gaa gatgac tgg cga tct cgg cct tcc agg gct cct 1204 Thr Ser Leu His Glu Asp AspTrp Arg Ser Arg Pro Ser Arg Ala Pro 380 385 390 gcc ctc aca ccc atc agggat gag gag tgg aat cgc cac tcc cct cgg 1252 Ala Leu Thr Pro Ile Arg AspGlu Glu Trp Asn Arg His Ser Pro Arg 395 400 405 agt ccc aga aca tgg gagcag gaa ccc ctt caa gaa cag cca agg ggt 1300 Ser Pro Arg Thr Trp Glu GlnGlu Pro Leu Gln Glu Gln Pro Arg Gly 410 415 420 425 ggt tgg ggg tct gggcgg cct cgg gcc cgc tct gtg gat gct cta gat 1348 Gly Trp Gly Ser Gly ArgPro Arg Ala Arg Ser Val Asp Ala Leu Asp 430 435 440 gac atc aac cgg cctggc tcc act gaa tca gga agg tct tct ccc cca 1396 Asp Ile Asn Arg Pro GlySer Thr Glu Ser Gly Arg Ser Ser Pro Pro 445 450 455 agt agt gga cgg agaggg cgg gcc tat gca cct ccg aga agt cgc agc 1444 Ser Ser Gly Arg Arg GlyArg Ala Tyr Ala Pro Pro Arg Ser Arg Ser 460 465 470 cgg gat gac ctc tatgac ccc gac gat cct aga gac ttg cca cat tcc 1492 Arg Asp Asp Leu Tyr AspPro Asp Asp Pro Arg Asp Leu Pro His Ser 475 480 485 cga gat ccc cac tattat gat gat ttg agg tct agg gat cca cgt gct 1540 Arg Asp Pro His Tyr TyrAsp Asp Leu Arg Ser Arg Asp Pro Arg Ala 490 495 500 505 gac ccc aga tcccgt cag cga tcc cac gat cct cgg gat gct ggc ttc 1588 Asp Pro Arg Ser ArgGln Arg Ser His Asp Pro Arg Asp Ala Gly Phe 510 515 520 agg tca cgg gaccct cag tat gat ggg cga ctc tta gaa gag gct tta 1636 Arg Ser Arg Asp ProGln Tyr Asp Gly Arg Leu Leu Glu Glu Ala Leu 525 530 535 aag aaa aaa ggggct ggg gag aga aga cgc gtt tac agg gag gaa gaa 1684 Lys Lys Lys Gly AlaGly Glu Arg Arg Arg Val Tyr Arg Glu Glu Glu 540 545 550 gaa gaa gaa gaggag ggc cac tat ccc cca gca cct ccg cct tac tct 1732 Glu Glu Glu Glu GluGly His Tyr Pro Pro Ala Pro Pro Pro Tyr Ser 555 560 565 gag act gac tcgcag gcc tcg agg gag cgg agg atg aaa aag aat ttg 1780 Glu Thr Asp Ser GlnAla Ser Arg Glu Arg Arg Met Lys Lys Asn Leu 570 575 580 585 gcc ctg agtcgg gaa agt tta gtc gtc tga tcccacgttt tgttatgtag 1830 Ala Leu Ser ArgGlu Ser Leu Val Val * 590 cttttatact tttttaattg gaatattgat gaaactcttcaccaagccta ataaaa 1886 14 1829 DNA Mus musculus 14 gcaccgtcgc tgctagacggccgcg atg gcg ccg gcg gcc agc gcg tgt gct 52 Met Ala Pro Ala Ala Ser AlaCys Ala 1 5 ggg gcg cct ggc tcc cac ccg gcc acc acg atc ttc gtg tgt cttttt 100 Gly Ala Pro Gly Ser His Pro Ala Thr Thr Ile Phe Val Cys Leu Phe10 15 20 25 ctc atc att tac tgc cca gac cgt gcc agt gcc atc cag gtg accgtg 148 Leu Ile Ile Tyr Cys Pro Asp Arg Ala Ser Ala Ile Gln Val Thr Val30 35 40 cct gac ccc tac cac gta gtg atc ctg ttc cag cca gtg aca cta cac196 Pro Asp Pro Tyr His Val Val Ile Leu Phe Gln Pro Val Thr Leu His 4550 55 tgc acc tac cag atg agc aat acc ctc aca gcc cct atc gtg atc tgg244 Cys Thr Tyr Gln Met Ser Asn Thr Leu Thr Ala Pro Ile Val Ile Trp 6065 70 aag tat aag tcg ttc tgt cgg gac cgt gtt gcc gac gcc ttc tcc cct292 Lys Tyr Lys Ser Phe Cys Arg Asp Arg Val Ala Asp Ala Phe Ser Pro 7580 85 gcc agc gtg gac aac cag ctc aac gcc cag ctg gcg gct ggc aac ccc340 Ala Ser Val Asp Asn Gln Leu Asn Ala Gln Leu Ala Ala Gly Asn Pro 9095 100 105 ggc tac aac ccc tat gtg gag tgc cag gac agc gta cgc act gtcagg 388 Gly Tyr Asn Pro Tyr Val Glu Cys Gln Asp Ser Val Arg Thr Val Arg110 115 120 gtg gtg gcc acc aaa cag ggc aat gct gtg acc ctg gga gac tactac 436 Val Val Ala Thr Lys Gln Gly Asn Ala Val Thr Leu Gly Asp Tyr Tyr125 130 135 cag ggc agg aga atc acc atc aca gga aat gct ggc ctg acc ttcgag 484 Gln Gly Arg Arg Ile Thr Ile Thr Gly Asn Ala Gly Leu Thr Phe Glu140 145 150 cag acg gcc tgg gga gac agt gga gtg tat tac tgc tcc gtg gtctca 532 Gln Thr Ala Trp Gly Asp Ser Gly Val Tyr Tyr Cys Ser Val Val Ser155 160 165 gcc caa gat ctg gat ggg aac aac gag gcg tac gca gag ctc attgtc 580 Ala Gln Asp Leu Asp Gly Asn Asn Glu Ala Tyr Ala Glu Leu Ile Val170 175 180 185 ctt gat tgg ctc ttt gtg gtc gtg gtc tgc ctg gca agc ctcctc ttc 628 Leu Asp Trp Leu Phe Val Val Val Val Cys Leu Ala Ser Leu LeuPhe 190 195 200 ttc ctc ctc ctg ggc atc tgc tgg tgc cag tgc tgt ccc cacacc tgc 676 Phe Leu Leu Leu Gly Ile Cys Trp Cys Gln Cys Cys Pro His ThrCys 205 210 215 tgc tgc tat gtc aga tgt ccc tgc tgc cca gac aag tgc tgttgc cct 724 Cys Cys Tyr Val Arg Cys Pro Cys Cys Pro Asp Lys Cys Cys CysPro 220 225 230 gag gcc ctt tat gct gct ggc aaa gca gcc acc tca ggt gtgcca agc 772 Glu Ala Leu Tyr Ala Ala Gly Lys Ala Ala Thr Ser Gly Val ProSer 235 240 245 atc tat gcc ccc agc atc tat acc cac ctc tct cct gcc aagact ccg 820 Ile Tyr Ala Pro Ser Ile Tyr Thr His Leu Ser Pro Ala Lys ThrPro 250 255 260 265 cca cct ccg cct gcc atg att ccc atg cgt cct ccc tatggg tac cct 868 Pro Pro Pro Pro Ala Met Ile Pro Met Arg Pro Pro Tyr GlyTyr Pro 270 275 280 gga gac ttt gac agg acc agc tca gtt ggt ggc cac agctcc cag gtg 916 Gly Asp Phe Asp Arg Thr Ser Ser Val Gly Gly His Ser SerGln Val 285 290 295 ccc ctg ctg cgt gaa gtg gat ggg agc gta tct tca gaagta cga agt 964 Pro Leu Leu Arg Glu Val Asp Gly Ser Val Ser Ser Glu ValArg Ser 300 305 310 ggc tac agg atc cag gct aac cag caa gat gac tcc atgagg gtc cta 1012 Gly Tyr Arg Ile Gln Ala Asn Gln Gln Asp Asp Ser Met ArgVal Leu 315 320 325 tac tat atg gag aag gag cta gcc aac ttc gat cct tcccgg cct ggc 1060 Tyr Tyr Met Glu Lys Glu Leu Ala Asn Phe Asp Pro Ser ArgPro Gly 330 335 340 345 cct ccc aat ggc cga gtg gaa cgg gcc atg agt gaagta acc tcc ctc 1108 Pro Pro Asn Gly Arg Val Glu Arg Ala Met Ser Glu ValThr Ser Leu 350 355 360 cat gaa gat gac tgg cga tct cgg cct tcc agg gctcct gcc ctc aca 1156 His Glu Asp Asp Trp Arg Ser Arg Pro Ser Arg Ala ProAla Leu Thr 365 370 375 ccc atc agg gat gag gag tgg aat cgc cac tcc cctcgg agt ccc aga 1204 Pro Ile Arg Asp Glu Glu Trp Asn Arg His Ser Pro ArgSer Pro Arg 380 385 390 aca tgg gag cag gaa ccc ctt caa gaa cag cca aggggt ggt tgg ggg 1252 Thr Trp Glu Gln Glu Pro Leu Gln Glu Gln Pro Arg GlyGly Trp Gly 395 400 405 tct ggg cgg cct cgg gcc cgc tct gtg gat gct ctagat gac atc aac 1300 Ser Gly Arg Pro Arg Ala Arg Ser Val Asp Ala Leu AspAsp Ile Asn 410 415 420 425 cgg cct ggc tcc act gaa tca gga agg tct tctccc cca agt agt gga 1348 Arg Pro Gly Ser Thr Glu Ser Gly Arg Ser Ser ProPro Ser Ser Gly 430 435 440 cgg aga ggg cgg gcc tat gca cct ccg aga agtcgc agc cgg gat gac 1396 Arg Arg Gly Arg Ala Tyr Ala Pro Pro Arg Ser ArgSer Arg Asp Asp 445 450 455 ctc tat gac ccc gac gat cct aga gac ttg ccacat tcc cga gat ccc 1444 Leu Tyr Asp Pro Asp Asp Pro Arg Asp Leu Pro HisSer Arg Asp Pro 460 465 470 cac tat tat gat gat ttg agg tct agg gat ccacgt gct gac ccc aga 1492 His Tyr Tyr Asp Asp Leu Arg Ser Arg Asp Pro ArgAla Asp Pro Arg 475 480 485 tcc cgt cag cga tcc cac gat cct cgg gat gctggc ttc agg tca cgg 1540 Ser Arg Gln Arg Ser His Asp Pro Arg Asp Ala GlyPhe Arg Ser Arg 490 495 500 505 gac cct cag tat gat ggg cga ctc tta gaagag gct tta aag aaa aaa 1588 Asp Pro Gln Tyr Asp Gly Arg Leu Leu Glu GluAla Leu Lys Lys Lys 510 515 520 ggg gct ggg gag aga aga cgc gtt tac agggag gaa gaa gaa gaa gaa 1636 Gly Ala Gly Glu Arg Arg Arg Val Tyr Arg GluGlu Glu Glu Glu Glu 525 530 535 gag gag ggc cac tat ccc cca gca cct ccgcct tac tct gag act gac 1684 Glu Glu Gly His Tyr Pro Pro Ala Pro Pro ProTyr Ser Glu Thr Asp 540 545 550 tcg cag gcc tcg agg gag cgg agg atg aaaaag aat ttg gcc ctg agt 1732 Ser Gln Ala Ser Arg Glu Arg Arg Met Lys LysAsn Leu Ala Leu Ser 555 560 565 cgg gaa agt tta gtc gtc tga tcccacgttttgttatgtag cttttatact 1783 Arg Glu Ser Leu Val Val * 570 575 tttttaattggaatattgat gaaactcttc accaagccta ataaaa 1829 15 1682 DNA Mus musculus 15gcaccgtcgc tgctagacgg ccgcg atg gcg ccg gcg gcc agc gcg tgt gct 52 MetAla Pro Ala Ala Ser Ala Cys Ala 1 5 ggg gcg cct ggc tcc cac ccg gcc accacg atc ttc gtg tgt ctt ttt 100 Gly Ala Pro Gly Ser His Pro Ala Thr ThrIle Phe Val Cys Leu Phe 10 15 20 25 ctc atc att tac tgc cca gac cgt gccagt gcc atc cag gtg acc gtg 148 Leu Ile Ile Tyr Cys Pro Asp Arg Ala SerAla Ile Gln Val Thr Val 30 35 40 cct gac ccc tac cac gta gtg atc ctg ttccag cca gtg aca cta cac 196 Pro Asp Pro Tyr His Val Val Ile Leu Phe GlnPro Val Thr Leu His 45 50 55 tgc acc tac cag atg agc aat acc ctc aca gcccct atc gtg atc tgg 244 Cys Thr Tyr Gln Met Ser Asn Thr Leu Thr Ala ProIle Val Ile Trp 60 65 70 aag tat aag tcg ttc tgt cgg gac cgt gtt gcc gacgcc ttc tcc cct 292 Lys Tyr Lys Ser Phe Cys Arg Asp Arg Val Ala Asp AlaPhe Ser Pro 75 80 85 gcc agc gtg gac aac cag ctc aac gcc cag ctg gcg gctggc aac ccc 340 Ala Ser Val Asp Asn Gln Leu Asn Ala Gln Leu Ala Ala GlyAsn Pro 90 95 100 105 ggc tac aac ccc tat gtg gag tgc cag gac agc gtacgc act gtc agg 388 Gly Tyr Asn Pro Tyr Val Glu Cys Gln Asp Ser Val ArgThr Val Arg 110 115 120 gtg gtg gcc acc aaa cag ggc aat gct gtg acc ctggga gac tac tac 436 Val Val Ala Thr Lys Gln Gly Asn Ala Val Thr Leu GlyAsp Tyr Tyr 125 130 135 cag ggc agg aga atc acc atc aca gga aat gct ggcctg acc ttc gag 484 Gln Gly Arg Arg Ile Thr Ile Thr Gly Asn Ala Gly LeuThr Phe Glu 140 145 150 cag acg gcc tgg gga gac agt gga gtg tat tac tgctcc gtg gtc tca 532 Gln Thr Ala Trp Gly Asp Ser Gly Val Tyr Tyr Cys SerVal Val Ser 155 160 165 gcc caa gat ctg gat ggg aac aac gag gcg tac gcagag ctc att gtc 580 Ala Gln Asp Leu Asp Gly Asn Asn Glu Ala Tyr Ala GluLeu Ile Val 170 175 180 185 ctt gtt tat gct gct ggc aaa gca gcc acc tcaggt gtg cca agc atc 628 Leu Val Tyr Ala Ala Gly Lys Ala Ala Thr Ser GlyVal Pro Ser Ile 190 195 200 tat gcc ccc agc atc tat acc cac ctc tct cctgcc aag act ccg cca 676 Tyr Ala Pro Ser Ile Tyr Thr His Leu Ser Pro AlaLys Thr Pro Pro 205 210 215 cct ccg cct gcc atg att ccc atg cgt cct ccctat ggg tac cct gga 724 Pro Pro Pro Ala Met Ile Pro Met Arg Pro Pro TyrGly Tyr Pro Gly 220 225 230 gac ttt gac agg acc agc tca gtt ggt ggc cacagc tcc cag gtg ccc 772 Asp Phe Asp Arg Thr Ser Ser Val Gly Gly His SerSer Gln Val Pro 235 240 245 ctg ctg cgt gaa gtg gat ggg agc gta tct tcagaa gta cga agt ggc 820 Leu Leu Arg Glu Val Asp Gly Ser Val Ser Ser GluVal Arg Ser Gly 250 255 260 265 tac agg atc cag gct aac cag caa gat gactcc atg agg gtc cta tac 868 Tyr Arg Ile Gln Ala Asn Gln Gln Asp Asp SerMet Arg Val Leu Tyr 270 275 280 tat atg gag aag gag cta gcc aac ttc gatcct tcc cgg cct ggc cct 916 Tyr Met Glu Lys Glu Leu Ala Asn Phe Asp ProSer Arg Pro Gly Pro 285 290 295 ccc aat ggc cga gtg gaa cgg gcc atg agtgaa gta acc tcc ctc cat 964 Pro Asn Gly Arg Val Glu Arg Ala Met Ser GluVal Thr Ser Leu His 300 305 310 gaa gat gac tgg cga tct cgg cct tcc agggct cct gcc ctc aca ccc 1012 Glu Asp Asp Trp Arg Ser Arg Pro Ser Arg AlaPro Ala Leu Thr Pro 315 320 325 atc agg gat gag gag tgg aat cgc cac tcccct cgg agt ccc aga aca 1060 Ile Arg Asp Glu Glu Trp Asn Arg His Ser ProArg Ser Pro Arg Thr 330 335 340 345 tgg gag cag gaa ccc ctt caa gaa cagcca agg ggt ggt tgg ggg tct 1108 Trp Glu Gln Glu Pro Leu Gln Glu Gln ProArg Gly Gly Trp Gly Ser 350 355 360 ggg cgg cct cgg gcc cgc tct gtg gatgct cta gat gac atc aac cgg 1156 Gly Arg Pro Arg Ala Arg Ser Val Asp AlaLeu Asp Asp Ile Asn Arg 365 370 375 cct ggc tcc act gaa tca gga agg tcttct ccc cca agt agt gga cgg 1204 Pro Gly Ser Thr Glu Ser Gly Arg Ser SerPro Pro Ser Ser Gly Arg 380 385 390 aga ggg cgg gcc tat gca cct ccg agaagt cgc agc cgg gat gac ctc 1252 Arg Gly Arg Ala Tyr Ala Pro Pro Arg SerArg Ser Arg Asp Asp Leu 395 400 405 tat gac ccc gac gat cct aga gac ttgcca cat tcc cga gat ccc cac 1300 Tyr Asp Pro Asp Asp Pro Arg Asp Leu ProHis Ser Arg Asp Pro His 410 415 420 425 tat tat gat gat ttg agg tct agggat cca cgt gct gac ccc aga tcc 1348 Tyr Tyr Asp Asp Leu Arg Ser Arg AspPro Arg Ala Asp Pro Arg Ser 430 435 440 cgt cag cga tcc cac gat cct cgggat gct ggc ttc agg tca cgg gac 1396 Arg Gln Arg Ser His Asp Pro Arg AspAla Gly Phe Arg Ser Arg Asp 445 450 455 cct cag tat gat ggg cga ctc ttagaa gag gct tta aag aaa aaa ggg 1444 Pro Gln Tyr Asp Gly Arg Leu Leu GluGlu Ala Leu Lys Lys Lys Gly 460 465 470 gct ggg gag aga aga cgc gtt tacagg gag gaa gaa gaa gaa gaa gag 1492 Ala Gly Glu Arg Arg Arg Val Tyr ArgGlu Glu Glu Glu Glu Glu Glu 475 480 485 gag ggc cac tat ccc cca gca cctccg cct tac tct gag act gac tcg 1540 Glu Gly His Tyr Pro Pro Ala Pro ProPro Tyr Ser Glu Thr Asp Ser 490 495 500 505 cag gcc tcg agg gag cgg aggatg aaa aag aat ttg gcc ctg agt cgg 1588 Gln Ala Ser Arg Glu Arg Arg MetLys Lys Asn Leu Ala Leu Ser Arg 510 515 520 gaa agt tta gtc gtc tgatcccacgttt tgttatgtag cttttatact 1636 Glu Ser Leu Val Val * 525tttttaattg gaatattgat gaaactcttc accaagccta ataaaa 1682 16 594 PRT Musmusculus 16 Met Ala Pro Ala Ala Ser Ala Cys Ala Gly Ala Pro Gly Ser HisPro 1 5 10 15 Ala Thr Thr Ile Phe Val Cys Leu Phe Leu Ile Ile Tyr CysPro Asp 20 25 30 Arg Ala Ser Ala Ile Gln Val Thr Val Pro Asp Pro Tyr HisVal Val 35 40 45 Ile Leu Phe Gln Pro Val Thr Leu His Cys Thr Tyr Gln MetSer Asn 50 55 60 Thr Leu Thr Ala Pro Ile Val Ile Trp Lys Tyr Lys Ser PheCys Arg 65 70 75 80 Asp Arg Val Ala Asp Ala Phe Ser Pro Ala Ser Val AspAsn Gln Leu 85 90 95 Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly Tyr Asn ProTyr Val Glu 100 105 110 Cys Gln Asp Ser Val Arg Thr Val Arg Val Val AlaThr Lys Gln Gly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln GlyArg Arg Ile Thr Ile 130 135 140 Thr Gly Asn Ala Gly Leu Thr Phe Glu GlnThr Ala Trp Gly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val ValSer Ala Gln Asp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr Ala Glu LeuIle Val Leu Gly Arg Thr Ser Glu Ala 180 185 190 Pro Glu Leu Leu Pro GlyPhe Arg Ala Gly Pro Leu Glu Asp Trp Leu 195 200 205 Phe Val Val Val ValCys Leu Ala Ser Leu Leu Phe Phe Leu Leu Leu 210 215 220 Gly Ile Cys TrpCys Gln Cys Cys Pro His Thr Cys Cys Cys Tyr Val 225 230 235 240 Arg CysPro Cys Cys Pro Asp Lys Cys Cys Cys Pro Glu Ala Leu Tyr 245 250 255 AlaAla Gly Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala Pro 260 265 270Ser Ile Tyr Thr His Leu Ser Pro Ala Lys Thr Pro Pro Pro Pro Pro 275 280285 Ala Met Ile Pro Met Arg Pro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp 290295 300 Arg Thr Ser Ser Val Gly Gly His Ser Ser Gln Val Pro Leu Leu Arg305 310 315 320 Glu Val Asp Gly Ser Val Ser Ser Glu Val Arg Ser Gly TyrArg Ile 325 330 335 Gln Ala Asn Gln Gln Asp Asp Ser Met Arg Val Leu TyrTyr Met Glu 340 345 350 Lys Glu Leu Ala Asn Phe Asp Pro Ser Arg Pro GlyPro Pro Asn Gly 355 360 365 Arg Val Glu Arg Ala Met Ser Glu Val Thr SerLeu His Glu Asp Asp 370 375 380 Trp Arg Ser Arg Pro Ser Arg Ala Pro AlaLeu Thr Pro Ile Arg Asp 385 390 395 400 Glu Glu Trp Asn Arg His Ser ProArg Ser Pro Arg Thr Trp Glu Gln 405 410 415 Glu Pro Leu Gln Glu Gln ProArg Gly Gly Trp Gly Ser Gly Arg Pro 420 425 430 Arg Ala Arg Ser Val AspAla Leu Asp Asp Ile Asn Arg Pro Gly Ser 435 440 445 Thr Glu Ser Gly ArgSer Ser Pro Pro Ser Ser Gly Arg Arg Gly Arg 450 455 460 Ala Tyr Ala ProPro Arg Ser Arg Ser Arg Asp Asp Leu Tyr Asp Pro 465 470 475 480 Asp AspPro Arg Asp Leu Pro His Ser Arg Asp Pro His Tyr Tyr Asp 485 490 495 AspLeu Arg Ser Arg Asp Pro Arg Ala Asp Pro Arg Ser Arg Gln Arg 500 505 510Ser His Asp Pro Arg Asp Ala Gly Phe Arg Ser Arg Asp Pro Gln Tyr 515 520525 Asp Gly Arg Leu Leu Glu Glu Ala Leu Lys Lys Lys Gly Ala Gly Glu 530535 540 Arg Arg Arg Val Tyr Arg Glu Glu Glu Glu Glu Glu Glu Glu Gly His545 550 555 560 Tyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu Thr Asp Ser GlnAla Ser 565 570 575 Arg Glu Arg Arg Met Lys Lys Asn Leu Ala Leu Ser ArgGlu Ser Leu 580 585 590 Val Val 17 575 PRT Mus musculus 17 Met Ala ProAla Ala Ser Ala Cys Ala Gly Ala Pro Gly Ser His Pro 1 5 10 15 Ala ThrThr Ile Phe Val Cys Leu Phe Leu Ile Ile Tyr Cys Pro Asp 20 25 30 Arg AlaSer Ala Ile Gln Val Thr Val Pro Asp Pro Tyr His Val Val 35 40 45 Ile LeuPhe Gln Pro Val Thr Leu His Cys Thr Tyr Gln Met Ser Asn 50 55 60 Thr LeuThr Ala Pro Ile Val Ile Trp Lys Tyr Lys Ser Phe Cys Arg 65 70 75 80 AspArg Val Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gln Leu 85 90 95 AsnAla Gln Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val Glu 100 105 110Cys Gln Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys Gln Gly 115 120125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly Arg Arg Ile Thr Ile 130135 140 Thr Gly Asn Ala Gly Leu Thr Phe Glu Gln Thr Ala Trp Gly Asp Ser145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gln Asp Leu AspGly Asn 165 170 175 Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Asp Trp LeuPhe Val Val 180 185 190 Val Val Cys Leu Ala Ser Leu Leu Phe Phe Leu LeuLeu Gly Ile Cys 195 200 205 Trp Cys Gln Cys Cys Pro His Thr Cys Cys CysTyr Val Arg Cys Pro 210 215 220 Cys Cys Pro Asp Lys Cys Cys Cys Pro GluAla Leu Tyr Ala Ala Gly 225 230 235 240 Lys Ala Ala Thr Ser Gly Val ProSer Ile Tyr Ala Pro Ser Ile Tyr 245 250 255 Thr His Leu Ser Pro Ala LysThr Pro Pro Pro Pro Pro Ala Met Ile 260 265 270 Pro Met Arg Pro Pro TyrGly Tyr Pro Gly Asp Phe Asp Arg Thr Ser 275 280 285 Ser Val Gly Gly HisSer Ser Gln Val Pro Leu Leu Arg Glu Val Asp 290 295 300 Gly Ser Val SerSer Glu Val Arg Ser Gly Tyr Arg Ile Gln Ala Asn 305 310 315 320 Gln GlnAsp Asp Ser Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu 325 330 335 AlaAsn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Val Glu 340 345 350Arg Ala Met Ser Glu Val Thr Ser Leu His Glu Asp Asp Trp Arg Ser 355 360365 Arg Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp Glu Glu Trp 370375 380 Asn Arg His Ser Pro Arg Ser Pro Arg Thr Trp Glu Gln Glu Pro Leu385 390 395 400 Gln Glu Gln Pro Arg Gly Gly Trp Gly Ser Gly Arg Pro ArgAla Arg 405 410 415 Ser Val Asp Ala Leu Asp Asp Ile Asn Arg Pro Gly SerThr Glu Ser 420 425 430 Gly Arg Ser Ser Pro Pro Ser Ser Gly Arg Arg GlyArg Ala Tyr Ala 435 440 445 Pro Pro Arg Ser Arg Ser Arg Asp Asp Leu TyrAsp Pro Asp Asp Pro 450 455 460 Arg Asp Leu Pro His Ser Arg Asp Pro HisTyr Tyr Asp Asp Leu Arg 465 470 475 480 Ser Arg Asp Pro Arg Ala Asp ProArg Ser Arg Gln Arg Ser His Asp 485 490 495 Pro Arg Asp Ala Gly Phe ArgSer Arg Asp Pro Gln Tyr Asp Gly Arg 500 505 510 Leu Leu Glu Glu Ala LeuLys Lys Lys Gly Ala Gly Glu Arg Arg Arg 515 520 525 Val Tyr Arg Glu GluGlu Glu Glu Glu Glu Glu Gly His Tyr Pro Pro 530 535 540 Ala Pro Pro ProTyr Ser Glu Thr Asp Ser Gln Ala Ser Arg Glu Arg 545 550 555 560 Arg MetLys Lys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val Val 565 570 575 18 526PRT Mus musculus 18 Met Ala Pro Ala Ala Ser Ala Cys Ala Gly Ala Pro GlySer His Pro 1 5 10 15 Ala Thr Thr Ile Phe Val Cys Leu Phe Leu Ile IleTyr Cys Pro Asp 20 25 30 Arg Ala Ser Ala Ile Gln Val Thr Val Pro Asp ProTyr His Val Val 35 40 45 Ile Leu Phe Gln Pro Val Thr Leu His Cys Thr TyrGln Met Ser Asn 50 55 60 Thr Leu Thr Ala Pro Ile Val Ile Trp Lys Tyr LysSer Phe Cys Arg 65 70 75 80 Asp Arg Val Ala Asp Ala Phe Ser Pro Ala SerVal Asp Asn Gln Leu 85 90 95 Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly TyrAsn Pro Tyr Val Glu 100 105 110 Cys Gln Asp Ser Val Arg Thr Val Arg ValVal Ala Thr Lys Gln Gly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr TyrGln Gly Arg Arg Ile Thr Ile 130 135 140 Thr Gly Asn Ala Gly Leu Thr PheGlu Gln Thr Ala Trp Gly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys SerVal Val Ser Ala Gln Asp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr AlaGlu Leu Ile Val Leu Val Tyr Ala Ala Gly Lys 180 185 190 Ala Ala Thr SerGly Val Pro Ser Ile Tyr Ala Pro Ser Ile Tyr Thr 195 200 205 His Leu SerPro Ala Lys Thr Pro Pro Pro Pro Pro Ala Met Ile Pro 210 215 220 Met ArgPro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp Arg Thr Ser Ser 225 230 235 240Val Gly Gly His Ser Ser Gln Val Pro Leu Leu Arg Glu Val Asp Gly 245 250255 Ser Val Ser Ser Glu Val Arg Ser Gly Tyr Arg Ile Gln Ala Asn Gln 260265 270 Gln Asp Asp Ser Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu Ala275 280 285 Asn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Val GluArg 290 295 300 Ala Met Ser Glu Val Thr Ser Leu His Glu Asp Asp Trp ArgSer Arg 305 310 315 320 Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg AspGlu Glu Trp Asn 325 330 335 Arg His Ser Pro Arg Ser Pro Arg Thr Trp GluGln Glu Pro Leu Gln 340 345 350 Glu Gln Pro Arg Gly Gly Trp Gly Ser GlyArg Pro Arg Ala Arg Ser 355 360 365 Val Asp Ala Leu Asp Asp Ile Asn ArgPro Gly Ser Thr Glu Ser Gly 370 375 380 Arg Ser Ser Pro Pro Ser Ser GlyArg Arg Gly Arg Ala Tyr Ala Pro 385 390 395 400 Pro Arg Ser Arg Ser ArgAsp Asp Leu Tyr Asp Pro Asp Asp Pro Arg 405 410 415 Asp Leu Pro His SerArg Asp Pro His Tyr Tyr Asp Asp Leu Arg Ser 420 425 430 Arg Asp Pro ArgAla Asp Pro Arg Ser Arg Gln Arg Ser His Asp Pro 435 440 445 Arg Asp AlaGly Phe Arg Ser Arg Asp Pro Gln Tyr Asp Gly Arg Leu 450 455 460 Leu GluGlu Ala Leu Lys Lys Lys Gly Ala Gly Glu Arg Arg Arg Val 465 470 475 480Tyr Arg Glu Glu Glu Glu Glu Glu Glu Glu Gly His Tyr Pro Pro Ala 485 490495 Pro Pro Pro Tyr Ser Glu Thr Asp Ser Gln Ala Ser Arg Glu Arg Arg 500505 510 Met Lys Lys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val Val 515 520525 19 22976 DNA Homo sapiens exon 1898..2253 exon 3437..3781 exon12065..12184 exon 15045..15101 exon 15666..15812 exon 19479..19652 exon19799..19858 exon 19956..20087 exon 20229..20854 exon 20944..21094Misc_Feature 19956..19958 Potential variant splicing site AAG 19aacagtttgg cagttcctca aaaggttaaa aatagaacta ccaagtcacc cagcaattcc 60attcttaggc atatattcaa aagaaatgaa agcagatatt tgtacaccag tgttcacagc 120tgcactattt acaatagtca aaaggtagaa acaacctagg tccatccaca aatgaatgga 180taaataaaac gtagcatata catacaatgg tacactagtc cgctgtaaaa agaaattttg 240atcttactgc atgctacatg gcttcgacat actacaacat ggatggacct tgaaaacatt 300attctttgtg aaataaacta gacacaggac aaatgttaga cgattccact tatatgaggc 360acctagaatg ggcaatttgg taagcaaagt agaatagaaa ttactagggg cacaggtagc 420agggaatggg gagttactgt ttaatggtca cagagtttat gttggggatg atgaaacagt 480ttcggggata aagagtggtg attggtacac gacattgtga atatacttaa tgccactgaa 540ttttacactt gaagtggtta aagcgataaa tattatagtt tgcatatttt atcataaaaa 600tattttttta aacgatgaag ggacgtgaac gggttgaaat tttataaaaa gtggccaggg 660aaggtgtcac tgcaatggtg tcctacagga ggaggaagat catgtggaca tctgcgggaa 720gggtgttctg gcagagggag tagcacgggc gatggctctg aggactgtga gaagtatagt 780tggaaacagc gaggaggcca gggtgtccga agctgagtaa gccagagaga gtgggaggag 840gtgagataag agggggaagg tcagtttctg ctgagagtga ggaggagcca caggagggct 900gtgagcaggt ggacgtgatc tggcttgagt tttaacaggg ccagtagaac aaagcacgcc 960tgggtaccga aaccagccac tggccagttg gcaacctggg ggagtctaac gcgaggaagc 1020gcccagggtt cccccaggat gcgctttccc tcgccgccac ctggagacag cagagtcacg 1080cccagcgctg cgcaggctga tcgccgcgcc gcgcccccgc cctcggtcgc aggtggctcg 1140ttccgggaat tcctaagcgg aaaccggtcc caagccccgc gccttcgctc ggccccttta 1200agagccagaa tttccggagg gctgacccgg gggctaggga tgcccagggg ccgaaccaca 1260agttgggaac gggtggggga ggtggcgaaa acttccgaag tggaattcca acttttcctg 1320gccctgattc cccttgggca tccctgaggg ggcagagctt cccttccggg gactttagag 1380ggttcctcag gtcatctaac tgggagacac aggaggcccg aagcgccccc cctccacccg 1440gtccggagga accccagtgg aagtggagaa gtcaggcgcc accaacaagc ctctcccagc 1500caggactttg cttagactcg ctcctcccgg cagggcgcac ctaggcgggt ccatcgccag 1560ccggggagag gggtttgggc agggagggaa caggtgcgcg gcgggacccg ccctatctca 1620acaggtgaat cgctccaagt gggtctcggt tgcatggatc tcggtgcgct tggtttggcc 1680ggagcagatg ggggccggaa gggacctgtg gtccgcaggc gccctcccag cgggccagtc 1740acttggttcg ggccctgggg gacggagcgc acctgggtca gcccacttcc ggggagggag 1800gcagaggaac ccctccccgc cgctcacccc taagcccagc cctcggctcc cacccttgtg 1860tacctgggcc gaaccattca ccggagcgcg cagcgggtgg agtgtggctc ggaggaccgc 1920ggcgggtcaa gcacctttct cccccatatc tgaaagcatg ccctttgtcc acgtcgttta 1980cgctcattaa aacttccaga atgcaacagg acggacttgg agtagggaca aggaacggaa 2040gtgggaaggg gaggagcgtg cacccctcct ggccttggtg cgcgccgcgc cccctaaggt 2100actttggaag ggacgcgcgg gccagacgcg cccagacggc cgcgatggcg ctgttggccg 2160gcgggctctc cagagggctg ggctcccacc cggccgccgc aggccgggac gcggtcgtct 2220tcgtgtggct tctgcttagc acctggtgca caggtacggg gcacggggcc tctgacgctg 2280cggaacgccg gagggaactg tagaggggga tggatggagt tggaggcggc gggaagcggg 2340aagcgggggt ctcagaggct gggaccttcc gatcccctgg gtcttgggcg atctgttgcg 2400cgcgggagtg agaggaattc cccatttgtg ccggggagcg ctccccgcgc ccttatctgg 2460aagatagcag gaagtgaaac tccctggacg gtgagacccg gagcggcagg gagaatggaa 2520ctctttgtgg ggagggagtg gaagaccgcc cgatctctgg gaaaagaaaa gccgggatgg 2580gacttgggcg cacccgggga tttctaagtt ttggagtaac ggggagaggg cacgggaggg 2640ctggatcaga cgcttcctag agggacagag acgaaggaac aatgcctagg cctcgggtgg 2700gtgtgggact ggggactccc catcccccgc accccaccca cctcccgcgg gctccggatt 2760atacgtgcgt aagagtctgg tgggatggat ttacggactt gaaaccgact tctgctggca 2820ggctttcacc tggatgggat atttgggtgg tgatgaggtc tttcccgaga cacttttggt 2880tcagtcattt gaaatgactt tagagtaggg tgaggtggtg ggaggctgat ggagatattg 2940tgggggcttt agtccctcca tggcaaagca gttcaggcaa acaactccat ggttttccct 3000ccaaattcaa aaggccccgg gtaacctgga atccttcgta gtcggttttg aagtggggcc 3060ttgggcgctg ggggcatcaa catggccatc tgggcttgcc tgcccaggcc acacagaggc 3120cccttgttgt gggtgaatgg caaagggaag aggggactgg tgtggttcag aggccacagg 3180ctgggaagag ggatggcggg cgagtccaag gaaactggcc gtgtcaccgt gcacctgcca 3240cttcagcccc acgggtctat aaaatgggca tgattatcgt ggctacctca ctggtcctgg 3300caattaagga acaatgtgtg ccaggcactc tgtaaaccac atacttgcga gtgtcaagct 3360ggtgacaggt ggcgttcctg ttgaagcacc tccctgagct cacagcaacc cttgctgtct 3420ctcctcttgc cctcagctcc tgccagggcc atccaggtga ccgtgtccaa cccctaccac 3480gtggtgatcc tcttccagcc tgtgaccctg ccctgtacct accagatgac ctcgaccccc 3540acgcaaccca tcgtcatctg gaagtacaag tctttctgcc gggaccgcat cgccgatgcc 3600ttctccccgg ccagcgtcga caaccagctc aatgcccagc tggcagccgg gaacccaggc 3660tacaacccct acgttgagtg ccaggacagc gtgcgcaccg tcagggtcgt ggccaccaag 3720cagggcaacg ctgtgaccct gggagattac taccagggcc ggaggattac catcaccgga 3780agtatgttgg gcagggcagg gggatgaggc tgggcttgcc cgggtggtgg gactggcgtc 3840cttgtgcggg acctggagtc cccatctgaa agctcttgag tgccagtgtc tgaaaggacc 3900attgaaggga gcaattcttt tttttttttt ttttgaagat ggagtcttgc tctggactcc 3960aggctggagt gcagtggtgc gatctcagct cactgcaacc tccacctccc aggttcaagc 4020aattctcttg cctcagcctc ccgagtagct gggactccag gtgcgtgcca ccacgcccag 4080ttaatttttg tatttttagt agagatgggg tttcaccatg ttggccaggc tggtctcaaa 4140ctcctgacct caaatgatct gcccgccttg gcctcgcaaa gtgctgagag acaccatacc 4200cagcctaaag ggagcgattc tattctacta ttcttccttc tgctaatcct tccattcttt 4260aatttaataa cgaagatttt ttgagtacct gtcatatacc aggtgctgtt ctgggccctg 4320ggaatacagc tgttaacaaa atcatcaaac cacttccctc gtggagccca cattgcagtg 4380agagagacaa acacgacaca cactctcaag tccttgaaga taaagaaaac tgggtaacgg 4440agagaagagg ccagggtttg ttctataatc attaataaca cgagcagtaa gaagtaaaat 4500ttatctaagt aacaacttat aaagggtcta ctgtgtgcta agctctcatc caggttccca 4560aggattaact cagaccacac agtaattgaa tagattctat cattgtcatc ttacagaggc 4620ccagagagag aaagtgactt gcctagtgtc atagctggta acggggctgg gattctaact 4680cagccacttt gggtctagtg gccaagctcc taatcccttt gcttgcctag ggtggtccgc 4740agaggactca cagaggagat ggcaggagtg aactgcaggg gcaagagagc ttaatggaga 4800aagcctgtga catgccagga actgcacaca tattctccca ttgagtcctc tcctctaccc 4860tcctgacagc tgaggcacag agaggttacc ttgttcaaat gggtgcatag gaagtcaaag 4920tctggagctg gggtttgaac ccaggcagcc ctgagaacct tgttcttttt ttttgagacg 4980gagtctcgct ctgtcgccca ggctggagtg cagtggcggg atctcggctc actgcaactc 5040cgcctcccgg gttcacgcca ttctcctgcc tcagcctccc aagtagctgg gactacaggc 5100gcccgccact acgcccggct aattttttgt atttttagta gagacggggt ttcaccgttt 5160tagccgggat ggtctcgatc tcctgacctc gtgatccgcc cgcctcggcc tcccaaagtg 5220ctgggattac aggcgtgagc caccgcgccc ggccccttgt tcttaactgt aatgctgcct 5280cctgatagga tgtgcctgtt gggactaagt aaggggcagt cattcattca ttcatttggt 5340atttatcaag catcgactat gtgtcgttgg tgctggggat agaggtgatt gggatggctg 5400aagtttctgt cgtcaaggag atgacattct ggtggagtga gactggcagt aaataagcag 5460ataaagaaag agtatgagaa tttcaaagtc tgggcacggt ggctcacgtc tgtaatctca 5520gcactttggg aggccaaggt gggtggatca cctgaggtca ggagttccag accagcctgg 5580ccaacatggt gaaaccccgt ctctactaaa aatacaaaga ttagccaggc atggtggcac 5640atgcctgtaa tcccagctac tcaggaggct gaggcatgag aatcgcttga acccaggagg 5700cagaggttgc agtgagctga gatcgcacca ctgtactgca gtctgggcga cagagtgaga 5760ctctgtctca aaaaaaaaaa aaaaaaaaaa gactccgtca aggtataaga atgtcagaga 5820gtactaagtg ttgcaaagaa aataacacca ggctgggtgc attggctcat gcctgtaaat 5880ttcagcactt tgggaggcca aggcaggagg atcacttgag cctaggagtt tgagaccagc 5940ctggacaaca aaatgagacc ccatgtctac aaaaatttta aaaatttaaa aattagctgg 6000gcatggtggc atgtgcctgt ggtcccggct gctcaggagg ctgaggtggg aggattgctt 6060gggcttgaga ggtcaaggct tcagtgagtc atgatcgtgc cactgcattc cagcctgggt 6120gacagagtga gaccctgtct tgaaatgaaa agaaaatagg ctgggcgcag tggctcacac 6180ctgtaatccc agcactttgg gaggccgagg tgggtggatc acctgaggtc aggagatcga 6240gaccagcctg gccaacatgg tgaaatccca tctctactaa aaatacaaaa tttagccggg 6300cgtggtggtg ggcgcctgta atcccagcta ctcgggaggc tgaggcagga gaatcgcttg 6360aacctgggag gcgaaggttg cggtgcgcca agattgcgcc actgcactct agcctgggaa 6420acagtgagac tccgtcttaa aaaaaaaaga aaaaagaaaa tagcactggg tgatgtgcta 6480catggaatga cttgggctgt gaatatgatt tgaggagggc ctgggcctgg gccttacaga 6540acctagaagg cagagaggaa ggggaggggc agggtgccag ggatgaaggc tcacgtacct 6600catgtcttag tgtgtgttca ctgtcttaaa caagaattta aagttgggca tggggcagag 6660cggggaaggg agcatccctt tgcagacccc aagaagccag gaactggagc acattctgct 6720agaggatcga tgggaagcag ggttccaggg gctgagccta tgtcagtcct gtttcagagg 6780aggcaccagg cttgcttgcc ctgaatttct gtgggcagct cagccatgag catcctactg 6840ttattgaggt cacagggctg cttaggcccc ctcctctcta acccagggat tgtgcctgcc 6900tggaccaggc gtgactgcta agcttctgcc aggacaagcc aaatactgag ggtgcttcct 6960ctgctggacg caaaagtcca ggatgacccc ccaggctctg tctcggggaa ggggccctgc 7020atgctccagg ggcctcacag gcctgggtct ttcaaaccac ccccacctgg gcctgtgttt 7080gatcaaggcc ctgagtgtaa acatccattg tgtgtgtcct ttcaggaaat cccatagcca 7140taggagcttc ctctgtttca gctttgagga tggggaaaag tggactcccc gtggtgttcc 7200tagggtcacc cactgtgctg gggtttttct gttgttgttg ttttttttct gttgcccagg 7260ctggagtgca gtggtgcaat ctcagctcac tgcaacctct gcctcgcaag ttcaagtgat 7320tctccgcctc agcctcctga gtagctggga ttacaggtgc acaccaccac acctggctaa 7380tttttgtatc tttttggtag agatgggatt tcgccatgtt ggccaggctg gtctcaaact 7440cctgacctca ggtgatctgc ctgccttggc ctcccaaagt tctgggatta cagatgtgag 7500ccaccatgcc cggcctatcc tggtttcaaa agtgaaaata gtcctggata aggtagaagg 7560ctgtccactc caggcatccc tccggtccgg tggctcattc cctgctttgt ccttccatgc 7620tttgggtgat ggaccagcac ctggacagga ggccctgttc cacctcctcg ggctccttgg 7680ggtccaagtg cccccacctc cagctgcact gcagcagaga gcccatggga cctctgaaat 7740catgaaggtc acctttgcgg tgtataaaga aggaaccaga ggttggagat gtggaggagg 7800cctggctgct gttcccactg gagacctggc atcttctccc cgacctaaaa caatgaaagc 7860agtgctcagc ccggatgaga tcacggccag cccaagacca ggaacagggt acgccctgca 7920ggaagaaggt gtgcccagac cttaggatgg atcaaaagaa gccggaaaac tatatttttt 7980gtgagttttg aaaatgtcag acaggtcaaa caaaacacag tgaggtccag cctcggccta 8040caagatgcca gatttcaacc cctggcctat atgatctgtt tgccatggca ggcggttcct 8100gtccacctct tttgtttata gcagggacca gctcttgagc tccagtgttg aagaggcacg 8160gtcagggtct gatctgaaga cactggtggc tcatgcctgt aatcccagca cttcaggagg 8220ccgaggcagg aggattgctt gaggacagga gctgggagac cagcctgggc aacacagtga 8280gacccagaga ctacaaaaaa ataaatttag cggggcatga tggcacaccc tgctactctg 8340gagatgggaa gattgcttga gcctaggagt tcgaagctgc agtgacccat gatcgcacca 8400ctgcactcca gcctgggcga ccaagctagg ccctctcaaa aaagatacag gtggaaaaat 8460gatggacgaa gagggcattg tggcaaacct ggggatttag gagaacctag tttggaattc 8520tatgaggatt caatgaaaga atgtgtgtag aggggcccag cacatagtaa gagctcaata 8580aacggtgggg gctaggggcg gtggctcatg cctgtaatcc cagcactttg ggaggctgag 8640gcaggtggat cacttgagcc ctggagttca agatcaacct ggacaacaaa gcaagatccc 8700atctcaaaat taaaaaacaa caccaacaac aaaaaaacag tggcttagat gcctgatcat 8760tagggtaagt cgtgtcctca accccttcac atctgctctg aaggtcacca tatccggaag 8820ccttccctgg cctccttgtt taaaatggca cagcccccac tccacgcctg gcactctctg 8880ctgtccctga ttcgttttct ccatacagct tatctttgtc tgatatgtga catagttaac 8940attttatatt tgtctttctt tcctagttag aatctgaact ctagaagggc aagggcaagg 9000atttataact caaaggttcc gggcttaggc ctcttttata ttcttgattt tgaggttaat 9060taagagctca ggcctagcga ggtggctcat gcctggaatc ccagcacttt gggaggccca 9120ggcgggcaga tcacttgagg tcaggagttc cagacctgcc tggccaacac agtgaaaaac 9180ctgtctctac taaaaataca aaaattagcc agttatgttg gcaggcgcct ataatcccag 9240ctactcaaga ggctgaggca ggagaatcgc ttgaacccag gaggcagagg ctgcagtgag 9300ccaagatcgt gccactgcac tccagcctgg gcaacagagc gagactccat ctcaaaaaaa 9360aaaaaaaaat taagagctca aagagtttgt tttcataggc agcagaatga gaaaagttta 9420caaaatagtt taaatgacaa taaagtcatt atagattaac ataaataaaa taccttttat 9480gaaaaaaata atcattttct gaaatcagac aaaacattgt gaatgagaag gtggcatggt 9540tttatttttt tgcaagtctc cgaagcctgg ctggatagaa gagcctggct tctcagagct 9600gcttcagtct gttgtgatat ctattgtatg tcacgtagcc tctggaaaac tccacagtta 9660gtattgttgg gaaaataact ttgacctcag gatctcctga aaacgtcttg gggaacccca 9720gggtctagag gctgcagttt gagaactgtt gctgtggtat cccaggtgtc tcaaatactg 9780cctagaacat aggtggtact cagtaattat tgttgaagga tgaatgaatg aatgaatgaa 9840tgaatgaatg aaagaaagaa agaaatgtgt ctttgaatcc agccatgtgc ccagaatgat 9900gagacagatg acaaaagcta agggacttta gcatgaggag agggggttcg tttccttttt 9960tttctttttt ttttgagatg gagtctcact ctactgccca ggctagagtg cagtggtgca 10020atctcagctc actgcaatct ctgcctcctg agttcaagca attctcctgc ctcagcctcc 10080agggtagctg ggactacagg tgcgtgccac catgcctagc taatttttta catttttggt 10140agagatgggg ttttaccatg ttggccgggc tggtctggaa ctcctgacct caagtgatcc 10200acctgcctca gcctcccaaa gtgttaggat tacaggtgtg agccaccatg tccggccaag 10260agggtgttca tttctgctcc ttgccaggta ttgtgtcagg cactggggac ccagcagtgg 10320ctgagacaga cagggctctg cctcacggag cccacatttt caccaggcaa aggatggtcg 10380gcccctaagc tgggagataa gacttcagca gttgggtggg ggagccgtgg gagaagccca 10440gcccacaggg ggacagtgca aatctagaac caaggcgatg gcaggggtga ggctggcacg 10500gtagctagag accacgtcgt gccaagggcc ttggggacca tgggactatg ggaccttagg 10560gaaggcgtct ggaatgctgt agccagacac tgttgcaagg aggatttttc tgtagacatg 10620aggccttcct tatgaagaaa gcaagggttc tttcattcct gggggtgcca ggtgctgtgg 10680actgcagcac gcgtggttgc tgccgtcaca gagctgtcat gcaggagggc agcgcgtcct 10740tgggaaggtg gcaggcaggt caggctagga ggaaagaggc cgggaagctg agggcatttc 10800ctgcccgaga tgcccaatgt agcctacttc tgtccccagt ggcttaaggc agagttgcct 10860ggtaggtgcc ctggtcccac cctggtgaaa ggctgaaggt atttaattag tgcctgagaa 10920gcagagagga aacaggatgt gccaaaacac tttgatggat ggtagagtta acaggctcct 10980tgcctgcagc tgcttcagac aagagcgtcc ccaagccctg ggcctgacct ggaatgtggg 11040gatggaaggg gagggggagg aaccaaggca ctgggagggt aagtctctct ctcccacata 11100gacacaccca ctccttatgg gtgcctgggc atctcctggt acctagaatc tggcctgttt 11160atctccacac ccatccctgg ggtctacact aggccctgtg ggtggcagtt cacatcaggg 11220gagttctgac tttggctctg agaggtggtt cagagatggc tgtaagttga gaagcacaga 11280ctgctgggtg tggtggttca cgcctgtaat cccagcactt tgggaggctg aggtgggggt 11340ggatcacctg aggtctggag ttcaaaacca acttggtcaa catggcgaaa ctccatctct 11400actaaaaatg caaaaattag ccaggtgtgg tggcaggtgc ctataatccc agctacatgg 11460gaggctgagg caggagaatt gcttgaatct gggaggcgaa gattgtagtg agccgagatt 11520agttcgcacc attgcatgcc agcctgggca acaagagtga aactccgatt caaacaaaaa 11580aaaaaaaaag ctgggcatgg tggagtgcct gtagtcctaa ctactcaggt gggaggattg 11640cttgagtcca ggaggttgaa gttgcagtgg gctataatta caccactgca ctccagccag 11700ggccacagag tgagaccctg tctctaaaga aagaaaaaaa aaaacaacct caggctccga 11760gggcaccatt actgctctat actgaagagc tgtgcagctt ttccagaccc gaaatgtcat 11820ccacaaaaca gaagtgataa tggtcctgcc tcacagactt cttgcagtag tccaggtgtt 11880tagaacgggg tgtaaaaggc cgtgtgccct tggtaggaat ctttgcatat gcatttgatc 11940atctgcagcc tgcccagccc actgcttgcc ccctcctggg tgtgctggga aggggtcttt 12000ggccctccag gggttaggtg ccccagcctc caaggtgccc tcacgccttt tcatcccgac 12060tcagatgctg acctgacctt tgaccagacg gcgtgggggg acagtggtgt gtattactgc 12120tccgtggtct cagcccagga cctccagggg aacaatgagg cctacgcaga gctcatcgtc 12180cttggtgagt gggcctggga agggggaggc atggcccttc cttttgtccg cttctgttct 12240gtctgccctc ccctgtgtcc gccctctgcc ctccagctta ccctctgggc tctgtcgcct 12300gctctgctct cccccaggct ctgccagtca cttaggctcc cctgtgccct gcaccccagg 12360cagggaccac tggcccacag tgcctccaat cacccaagcc aaactaagag aagagtggag 12420acaattggag actctgcctt ttcaaagtct catttttaaa aaaaatccag acttggggtc 12480cgggtgcggt agttcatgcc tgtaatccca gcactttggg aggccgaggc gggtggatca 12540cttgaggcca ggagttcgag actagcctgg ccaacgtggc aaaatcccgt ctctataaaa 12600aatataaaag ccaggcgtgg tggtgcacat gcctgtaatc ccagttactc agaaggctga 12660ggcatgagga ttgcttgaac ctgggaggca gaggatgcag taagccaaga tcaagccact 12720gcactccagc ctgggcgaca gagtgagact ctgtccaaaa aaaaaaaaaa tccagacgtg 12780gtcagagtcc atgggcagtg aatgaggaca gttgatggtg tgcaaaatcg acccacctct 12840tgctacatcc ccaaggcctc atctcacccg agtccctcgc caaagcacag cggttttgcc 12900gtgtgccctg ctgggatggc gctgcatggc acacacactg tgtaagtttg agtgcagctg 12960aaacgaagcc gattccagac acccaggggc agggcggggt gtccgtgtgg ctgggaggcc 13020tccttgtgtt agggggatgt tgccatcggc caggtgccct gctgtaagcc aacacatgga 13080gtcttgtatg acatgtgctc tgcatgagtg atgccgctgg gctgtacact gccatcttca 13140catgtgtgaa tgagcacgtg actggggggt acttgggctg caagacagag ttcatgtgtg 13200ggggatggaa cacgtgcacc agtgacccag gaacctctgc ctgttcttcg gtaaaatgca 13260ccatttgcat cagcagttcc caaaattagt ctccaggtct atttacactc taaaacatta 13320tcgagggtct ccaagagctt ttgtttgttt ctgtgggttt tatgtctatc tgttgcttaa 13380catattagga attaaaatgg ggagattttc cttttttttt tttttttttg agatggagtc 13440tcgttctgtc gcccaggctg gagtgcagtg gctcgatctc ggctcactgc aagcttcacc 13500tcctgggttc acgccattct cctgcctcag cctcccaggt agctgggact acaggcaccc 13560gccaccacac ccggctaatt ttttttgtat ttttagtaga gactgggttt caccatgtta 13620gccaggatgg tctcgatctc ctgacctcgt gatccaccca cctgggcctc ccaaagtgct 13680gggattacag gcatgagcca ctgcccggcc ttaaaatggg gagatttttc aagcccaaga 13740tacacaagga agactgggca acatggcaag accctgactc tacaaaaaat tttaaaatta 13800accaggcatg gtggcatgca cctgtgagcc cagcttcttg ggaggctgag gcaggagtat 13860cgcttgcacc caggaggtca aggctgcagt gagccgtgac tatgctactg cactctagca 13920tgagtgacag agaccctggc tcaagaaaca caaacacaca cacacacaca cacacgcata 13980tagtccatta ggcatcaggg cgatgatggc atcagggagc ctgggaaact ctactggaca 14040ttcatgggag aacaagtgaa aaaggcaaat aacatcttag tgttattcta aaatttcttc 14100ttttggcctt gtggacagga ccacgctttg agagctgtga ctgacatgcc tctgtcctgt 14160tgcgagggcc tatagtgcca agtgcatgag ctctggggag ggcttcgtgg gtgcagagct 14220gggcctgtgg aggcccctca gacacaacac tggtggggct cagagctcca ggggcactcg 14280agggaagaca agaaccggct ctgagatgcg tgaatgtgac agtgcatgag tagagatgga 14340gaccttgtgg gtcccagaac caggactgca tatgactttc atatgtgggt atttttgcct 14400tcatgggtcc cttcctgttt taaaaaaaat gtgtgattat gttgtcacaa agagtttatt 14460cctgtatatt gtgttaattt gtgttcagat ttgtaaagta aaattaaacc atttcagcca 14520ggtgtggtga cacatgcctg tagccctagc tacttacccc agaggctgag gtgggaggat 14580cgcctgagcc cacgaggttg aagctgcagt gagccatgat cacacccctg cactccagac 14640tgggcgacag agctgagatc ctatttcgtg ggccctaggt ccctgtgcct gctggaacag 14700gacatcccta tcaccgtggt tggagccctt tggggtgcta agacctatga atgagggaaa 14760cttagggtgc ccaagctgag gtagagccct cagaaccccc tgggatttgt attggagccc 14820tcgtggcata acacaggtgg attatgcaat gggagtttct tacctataag cacccacatg 14880tgggcgggtg gagggtagga gccatgcact agggcttcag cccccagccc cttcccgctt 14940cagggcacac cttgcacttg gccagcctgg agctgggctt tcgggggtgg cacagcctgg 15000gctggctctg gccagcataa tctgtttctc ttttgtccct ccagggagga cctcaggggt 15060ggctgagctc ttacctggtt ttcaggcggg gcccatagaa ggtacggggg gtggatcctg 15120agttgggctt ctcgggagct cccatacatc acctactgct tctgactcta gttagtatcc 15180ccttccccac taaaccctgc tcactgtgga cccctcacta acctggcctg actgtggctc 15240tgaggcatct agtggtctgg cgctgggcct aggctaggct gggctgagga gagcctgggg 15300tgcaggccag ggctctgtga ctggcacctg cggtgctctt gagggtgtgg cgtctgggca 15360gctggctctc tctttggtct gggggctgca gtctgtctcc ctctgtgcag gctgcctcgt 15420tttctgcctt gtgttttttg cacctggggg agggccgtaa ctggggaatg gccgggatgg 15480tagaatgggg agtgtgctgt gcccagcctc tggcacaaaa aatccagcca gggctgcagg 15540ttccttggtg agctttgcaa atcgtccccg acctcagtgc tggctccgca ccatgtaccc 15600ctgctgtgcc gttagccctg ttccctccca ggcctccggg ctcagggcct gttgtctttc 15660tgcagactgg ctcttcgtgg ttgtggtatg cctggctgcc ttcctcatct tcctcctcct 15720gggcatctgc tggtgccagt gctgcccgca cacttgctgc tgctacgtca ggtgcccctg 15780ctgcccagac aagtgctgct gccccgaggc ccgtaagtgt cccgctcatg gccaccctgg 15840tttgggcaac atcctgcatc caagggaagg aggtggccat ccacctgccc ccaggacagt 15900ggcgttggtc tggagggtgt gaatttagcc agtggggaga aagtaggctg aggagggtct 15960gctgtttaga ttgtcgttta cttcctccaa cttttagttt atttttattt atgttgttct 16020tttcttttgt aagtataatc catacacatg gtaaaaatgt ccaacagtac aagatactag 16080tcacatggaa gtaaagccct ctaaaaaaac caaatcttgg ctaggcgcag tgattacgcc 16140tgtaatccca gcactttggg aggccaagac gagtggatca cttgaggtca ggagttccag 16200atcagcctgg ccaacatggt aaaacccagt tctctactaa aaatacaaaa attagctggg 16260catggtggtg atcgcctgta atcccagcta ctcaggagac tgaggcatga gaatcgctta 16320aacccaagaa gtggaggttg cagtgagctg agatcacgcc actgcactcc agcctgggcg 16380acagagtgag actctgtctc aaaaaaaaaa gaaaaaaaaa tgttaagtga aaaagttaag 16440aaaccaaaca aggtttacaa cactacatga tttaagcaaa aaaaattttt tttgttttag 16500agaaagggtc tcattctgtc atccaggcag tgcagtgcga tcatagctct ctgcagcctc 16560aaactcccgg gttcaagcag tcctcccgcc tcagcctctg gagcagctgg gactgtaggc 16620acacaccacc atgcccagct aattttttga tttttgtttt ttgtagagac ggggtctcag 16680tatgttgccc agcctgatct caaactcctg gcctcaggtg atcctcccaa gtcagcctcc 16740ccaaagtgct gggattacag gcatgtgcca ccatgctggc caatttttaa aaattttctg 16800tagagacagg gtcttgctat gttgcccagg ctggtcttga actcttgacc tcaagtgatc 16860ctgcctcagg ctcccaaagt gatgggatta caggcatgaa ctaccacacc tggccttaaa 16920cttaagcaaa tttttttttt tttttggaga cagtttcact ctgtcgccca ggctggagta 16980aagtggcgtg atctctgctc actgcaacct ccgccccccg ggtttaagct attctcctgc 17040ctcagcctcc cgagtagctg ggatataggc gcctgccacc acgcctgact aatttttgta 17100tttttagtag agacggggtt ttgccatgtt ggccaggctg gtctcgaact cctgacctca 17160ggcagtccgc tcccccgcac ccctaccttg gcctcccaaa gtgttaggac tacaggtgtg 17220agccaccatg cctggccaaa tttaagcaaa tgtttgaaaa cacataccca caggaatgct 17280gcacatttta cccagctact atgtctaggg tcgtatctag cacaccagca tggctactgt 17340ggagagctgg gactggatgt gagatgagag ctaaagggga agtaagcaaa ccaagcaggg 17400gaaggtaaga gaagacagaa gacagagaga gagggaccta actctatgag aggagtcaga 17460catgtgcaat tgaaaaagac ttgctcctgt ctctcttctg tgaatgtttg tgaatatccc 17520aacgggacac tttcacagag gagctgattg acgtggtcac agccatcagc cttgggacac 17580cagaccacag tgtgtacact aagtggcact gatggacact tcagcatccc tctagctgct 17640gtcccgtttc ccctcctcgg ggaccacagc tgttgccagt ccttggtttc cttcaggagg 17700gtgtctgggt agaccagcct gtgtgcacac agtccaagat acatgaacag tgaagtgcca 17760ggcaatcctt gcaagcatgg gcaggtggag agctgaggcc tgcttgacac cttcctgctc 17820agaagcccag tgagcagttt ccctccctag ggctcagtgt catcccctat aaaatggggc 17880ttatggcaga gctcaccaca ctgggtgcat ctggggattt ggcgagctca tgtgcacacc 17940attgagcatg gggcccaacc tatataaaat attctacgtc tgtcagctgc tgggcactgc 18000cactatcagc ctcagtagtg actgagggac agggcaccag tcagagccct ggtgcacaca 18060gagtgacccc agagaagcag ccttccctct ctgagtcctg tttccttctg ttaggtcctg 18120acttcatggg ttgttgttag cattaaggaa gtcgctggct aattttatag tcattgaagt 18180cagtggtgtg caacctggtt cctcaaagga tcacttccct gaaaaaattc cactgctccc 18240tggaggctta tgcaggccat cccatcccct ccctcttgtt gtgttcagct gacagctttt 18300tgctcagtga gtaagtgtta ggtccatttc acagatgggc tgcaaccaag tttgcagtga 18360acccactaag accagagcta gggccaggac taaatgctgg tcccaatgcc acattcccct 18420gtccccacac cacatttcct ccatccggag accctgttac cccaacccag ggccccatta 18480actccctggc agaggccctg ttacatctgc tgctgccaca gcctccgccc acccttcagg 18540aggcagcagg tcccactgct gatgataaag ttgcaggctg cctgagctaa tgaaggggct 18600tcctctaggc tgtgcactta gtcttctgct tccaaaccaa atcagaggtg aggcaccctc 18660tctgggccca tctctctcct ccattttcct gttggggtcc cagggaggaa gccacttgcc 18720tagggcccag gaattttgca agcctcttgc cctagggagg aaggaaggga ggaggatctt 18780accttgaact gtcaagccta gagcctggtg gggcaggcag aaatgggtgc agtccatgag 18840ttagaaacac tagaggagac actttgctgc ttggccgggg caggcaagtt aattcccgag 18900gctcctgcca ctgcatctca atctggaagg tgaccaggtg ggcaggaccc acgtctccca 18960gatgactcat tttttctaga acaggggctt ggctgccaaa gaggatactt gatttcggct 19020tgtggggaca gtggtggacc cagcatctgg gctttatata aagggcagct ttgttgccct 19080gtaaacacac agaccatggg tggccacttc ttccagtaag ttagctgggg agttggaagt 19140ttaggtaaaa ccttttgatt gacaaatgtt ggcgaattac catgctgtta aatgaaacat 19200tgttctgcca ccctggggct gtgggtgcct gcgtgcaccc tctgaaaaat cacacaggaa 19260gtggggtggg gtctctgtga agctggtgtc ccccagcctc agggatgctg cagaaatgga 19320atgaggacca acagggactc agatgtccaa ggaagctcta cagcggagag gacggcttgg 19380gaaggaggtc caggcccagg tccctccgga acccaatggg tatggggcag cctggctcct 19440gcctcatccc ccttctcctg ttgattgtgt cctcacagtg tatgccgccg gcaaagcagc 19500cacctcaggt gttcccagca tttatgcccc cagcacctat gcccacctgt ctcccgccaa 19560gaccccaccc ccaccagcta tgattcccat gggccctgcc tacaacgggt accctggagg 19620ataccctgga gacgttgaca ggagtagctc aggtgaggcc gggggaagca ggaacagctg 19680gtgggagtgt gctgggcatc tggacactga ggggcagggg ctggaaggaa gagtgtcttg 19740ggagccgagg aggggctctg ctcctggtgc gcggccactg acagccactc tcccccagct 19800ggtggccaag gctcctatgt acccctgctt cgggacacgg acagcagtgt ggcctctggt 19860gagaatccat cgtcccgaag ttggatgtgc ctgtaaggga gaggggtggg ccaggatcca 19920tcctcccaaa ccgaccacca cccccctgtc cctagaagtc cgcagtggct acaggattca 19980ggccagccag caggacgact ccatgcgggt cctgtactac atggagaagg agctggccaa 20040cttcgaccct tctcgacctg gcccccccag tggccgtgtg gagcggggta agcaggagcc 20100ttggggtctg agggctttta aggtgggggg gtgaaacatg tctccctgat acctgccgca 20160gggactcttg gtgcaaaccc tggaccccgg gctcctccag cagtcagtga cacccccctt 20220ccctgcagcc atgagtgaag tcacctccct ccacgaggac gactggcgat ctcggccttc 20280ccggggccct gccctcaccc cgatccggga tgaggagtgg ggtggccact ccccccggag 20340tcccagggga tgggaccagg agcccgccag ggagcaggca ggcgggggct ggcgggccag 20400gcggccccgg gcccgctccg tggacgccct ggacgacctc accccgccga gcaccgccga 20460gtcagggagc aggtctccca cgagtaatgg tgggagaagc cgggcctaca tgcccccgcg 20520gagccgcagc cgggacgacc tctatgacca agacgactcg agggacttcc cacgctcccg 20580ggacccccac tacgacgact tcaggtctcg ggagcgccct cctgccgacc ccaggtccca 20640ccaccaccgt acccgggacc ctcgggacaa cggctccagg tccggggacc tcccctatga 20700tgggcggcta ctggaggagg ctgtgaggaa gaaggggtcg gaggagagga ggagacccca 20760caaggaggag gaggaagagg cctactaccc gcccgcgccg cccccgtact cggagaccga 20820ctcgcaggcg tcccgagagc gcaggctcaa gaaggtgagg gccgccctcc ctggcgtcca 20880gaccgtccct gggcccccag ccggtccccg cggctcatac ccttctttct ttctcccttg 20940cagaacttgg ccctgagtcg ggaaagttta gtcgtctgat ctgacgtttt ctacgtagct 21000tttgtatttt tttttttaat ttgaaggaac actgatgaag ccctgccata cccctcccga 21060gtctaataaa acgtataatc acaagctctg gagagaacca tttgttcggc cgcgcggggc 21120gggggaccgg ggctgctccc gtatgcgtct gtaaagcgcc gcgtcccggg ggcaccggag 21180tccggggccg ggaggaagag acccagcctg gcccggcccg cgcccgcgcc gccggccgga 21240gaacgtgccc cgcgcagccg ccgcccgcct gcgtgcgcgc cccggccccg cccaggcgtg 21300cgcatgcgcc ccggccctcc gccttcgcgc accgcaggct ggccgtccgg gacgcgcgcg 21360cgctcctctc cccttccagc ccatcccccc cagcccccca ccgacctact ttactgtctc 21420caaactcggg cagcccacct ggcccccgac gaccccagcc cctgcaccgg gtaccccgac 21480gttccatcca gacccgcgtt tcaccagggc ggcgcgcggc gacctcgcgc cccgcggagc 21540cccgggctcg cgcgcgcccg cccgcccccg gagacagacc agcgcgcgcg ccccgggccg 21600cctcccccca gcgcgcgtcc gccccggggc tcgcgccgcc gccgccgccg ccgccgcgcg 21660cgcgcagctc aagtaaagga ggaaaaaaaa aagggggaaa aatagaaagc ggcggcggct 21720gcagcagcga tccgccgccg gactgggcca agccgggcgg cggccgcgcg agccggcgat 21780ccagggcact ggcggcggcc agccagggcg ggccgtgttc aaaaaaaaaa gtcgcggcgg 21840cggcggctgc tcagggaagg aggcctgagg gccgcgtgca gcgggcgggc agctgggtgg 21900gctgggggcg gccgcgcggc gtcccggagc ctcgggccgc ccggagccgg cgggcgggcg 21960gaggcggagg cggcggcggc tgcagcggct gcaggagcgg cggcggctgc ggcggcggcg 22020gcggcatctc ctcctcacat gaccccactg tttgtccccg tgatcagcgc gagcggctcc 22080cgtatctcct ccgtcccctc ctgccgcgcg gcgtgagcgc cgggctcggg gcccccccgg 22140ccgcccgccc cctcccctcc ctccctcccc tcccctcccc tcccccccgg gccccgcgcc 22200ccccccgccc ccgccccccc catggacatg ctggacccgg gtctggatcc cgctgcctcg 22260gccaccgctg ctgccgccgc caggtaagat ccccggcccg gccgtgcccc cgcgccccgg 22320ccccggcccc ggccccgcgg cctgcaggcc ggggccgcca tgatcccgag cggccgcggg 22380cccggctcaa aatggaggcc gccggcgcgg gggggacctg gcgcctcccg cccccggccc 22440ccggcctcgg cggcgccccc ggcctcaggc gcggccgggt gggactgggg ccctgcagct 22500gggcgcgggg gcgggggcgc gggcgcgggc cgcgctgacc ctgctccctc ctgtgcccct 22560ggcagccacg acaagggacc cgaggcggag gagggcgtcg agctgcagga aggtgagtgc 22620ttgccgggcc ggccgcgccc ggggagggct gggggcgctc ggcgcggccc tgaccgtgcc 22680ccgaccctcc tcggccccag gcggggacgg cccaggagcg gaggagcaga cagcggtggc 22740catcaccagc gtccagcagg cggcgttcgg cgaccacaac atccagtacc agttccgcac 22800agagacaaat ggaggacagg tgagcggcgg gccgcgaggg cgaacgggcg ggcgggcggg 22860cgcgccggga aggctcggac ctggccccag cgccggcctc gccgctctgc cgccccctgc 22920aggtgacata ccgcgtagtc caggtgactg atggtcagct ggacggccag ggcgac 22976 2030 DNA Rattus norvegicus 20 gttacagaat tcgccgcgat ggcgccggcg 30 21 20DNA Rattus norvegicus 21 gccaggacag tgtacgcact 20 22 20 DNA Ratusnorvegicus 22 acctcaggtg tcccgagcat 20 23 20 DNA Rattus norvegicus 23gaagatgact ggcgatcgag 20 24 20 DNA Rattus norvegicus 24 acctctatgacccggacgat 20 25 20 DNA Rattus norvegicus 25 caccaccctg acagtgcgta 20 2620 DNA rattus norvegicus 26 ctgggggcat agatgctcgg 20 27 20 DNA Rattusnorvegicus 27 gccctggaag gcctcgatcg 20 28 20 DNA Rattus norvegicus 28caagtcccta ggatcgtccg 20 29 20 PRT Rattus norvegicus Misc_Feature169..188 Position in SEQID2 29 Ser Ala Gln Asp Leu Asp Gly Asn Asn GluAla Tyr Ala Glu Leu Ile 1 5 10 15 Val Leu Gly Arg 20 30 15 PRT Rattusnorvegicus Misc_Feature 556..570 Position in SEQID2 30 Glu Glu Gly GlnTyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu Thr 1 5 10 15 31 21 DNA Homosapiens 31 atgcaacagg acggacttgg a 21 32 28 DNA Homo sapiens 32tcagacgact aaactttccc gactcagg 28 33 20 DNA Homo sapiens 33 ctacaacccctacgttgagt 20 34 24 DNA Homo sapiens 34 tcgtgacctg acctttgacc agac 24 3524 DNA Homo sapiens 35 cctgagctac tcctgtcaac gtct 24 36 20 DNA Homosapiens 36 aggccgagat cgccagtcgt 20 37 27 DNA Homo sapiens 37 ctacatggatccagtcatgc cgaagat 27 38 28 DNA Homo sapiens 38 cgacaactcg agtcagttggtatcatgg 28 39 15 PRT Homo sapiens Misc_Feature 1..14 Identical to 5 ..18 in ref swissprot Q07021 39 Leu Arg Cys Val Pro Arg Val Leu Gly SerSer Val Ala Gly Tyr 1 5 10 15 40 16 PRT Homo sapiens Misc_Feature 2..15Identical to 268 .. 282 in ref swissprot Q07021 40 Cys Tyr Ile Thr PheLeu Glu Asp Leu Lys Ser Phe Val Lys Ser Gln 1 5 10 15 41 21721 DNA Homosapiens exon 1898..2253 exon 3438..3782 exon 12064..12183 exon15049..15105 exon 15670..15816 exon 19486..19659 exon 19806..19865 exon19963..20094 exon 20236..20864 exon 20954..21094 Misc_Feature 715diverging nucleotide, G in ref genbankAC002128 41 aacagtttgg cagttcctcaaaaggttaaa aatagaacta ccaagtcacc cagcaattcc 60 attcttaggc atatattcaaaagaaatgaa agcagatatt tgtacaccag tgttcacagc 120 tgcactattt acaatagtcaaaaggtagaa acaacctagg tccatccaca aatgaatgga 180 taaataaaac gtagcatatacatacaatgg tacactagtc cgctgtaaaa agaaattttg 240 atcttactgc atgctacatggcttcgacat actacaacat ggatggacct tgaaaacatt 300 attctttgtg aaataaactagacacaggac aaatgttaga cgattccact tatatgaggc 360 acctagaatg ggcaatttggtaagcaaagt agaatagaaa ttactagggg cacaggtagc 420 agggaatggg gagttactgtttaatggtca cagagtttat gttggggatg atgaaacagt 480 ttcggggata aagagtggtgactggtacac gacattgtga atatacttaa tgccactgaa 540 ttttacactt gaagtggttaaagcgataaa tattatagnt ttgcatattt tatcataaaa 600 atattttttt aaacgatgaagggacgtgaa cgggttgaaa ttttataaaa agtggccagg 660 gaaggtgtca ctgcaatggtgtcctacagg aggaggaaga tcatgtggac atctccggga 720 agggtgttct ggcagagggagtagcacggg cgatggctct gaggactgtg agaagtatag 780 ttggaaacag cgaggaggccagggtgtccg aagctgagta agccagagag agtgggagga 840 ggtgagataa gagggggaaggtcagtttct gctgagagtg aggaggagcc acaggagggc 900 tgtgagcagg tggacgtgatctggcttgag ttttaacagg gccagtagaa caaagcacgc 960 ctgggtaccg aaaccagccactggccagtt ggcaacctgg gggagtctaa cgcgaggaag 1020 cgcccagggt tcccccaggatgcgctttcc ctcgccgcca cctggagaca gcagagtcac 1080 gcccagcgct gcgcaggctgatcgccgcgc cgcgcccccg ccctcggtcg caggtggctc 1140 gttccgggaa ttcctaagcggaaaccggtc ccaagccccg cgccttcgct cggccccttt 1200 aagagccaga atttccggagggctgacccg gggctaggga tgcccagggg ccgaaccaca 1260 agttgggaac gggtgggggaggtggcgaaa acttccgaag tggaattcca acttttcctg 1320 gccctgattc cccttgggcatccctgangg ggcagagctt cccttccggg gactttagag 1380 ggttcctcag gtcatctaactgggagacac aggaggcccg aagcgccccc cctccacccg 1440 gtccggagga accccagtggaagtggagaa gtcaggcgcc accaacaagc ctctcccagc 1500 caggactttg cttagactcgctcctcccgg cagggcgcac ctaggcgggt ccatcgccag 1560 ccggggagag gggtttgggcagggagggaa caggtgcgcg gcgggacccg ccctatctca 1620 acaggtgaat cgctccaagtgggtctcggt tgcatggatc tcggtgcgct tggtttggcc 1680 ggagcagatg ggggccggaagggacctgtg gtccgcaggc gccctcccag cgggccagtc 1740 acttggttcg ggccctgggggacggagcgc acctgggtca gcccacttcc ggggagggag 1800 gcagaggaac ccctccccgccgctcacccc taagcccagc cctcggctcc cacccttgtg 1860 tacctgggcc gaaccattcaccggagcgcg cagcgggtgg agtgtggctc ggaggaccgc 1920 ggcgggtcaa gcacctttctcccccatatc tgaaagcatg ccctttgtcc acgtcgttta 1980 cgctcattaa aacttccagaatgcaacagg acggacttgg agtagggaca aggaacggaa 2040 gtgggaaggg gaggagcgtgcacccctcct ggccttggtg cgcgccgcgc cccctaaggt 2100 actttggaag ggacgcgcgggccagacgcg cccagacggc cgcgatggcg ctgttggccg 2160 gcgggctctc cagagggctgggctcccacc cggccgccgc aggccgggac gcggtcgtct 2220 tcgtgtggct tctgcttagcacctggtgca caggtacggg gcacggggcc tctgacgctg 2280 cggaacgccg gagggaactgtagaggggga tggatggagt tggaggcggc gggaagcggg 2340 aagcgggggt ctcagaggctgggaccttcc gatcccctgg gtcttgggcg atctgttgcg 2400 ncgcgggagt gagaggaattccccatttgt gccggggagc gctccccgcg cccttatctg 2460 gaagatagca ggaagtgaaactccctggac ggtgagaccc ggagcggcag ggagaatgga 2520 actctttgtg gggagggagtggaagaccgc ccgatctctg ggaaaagaaa agccgggatg 2580 ggacttgggc gcacccggggatttctaagt tttggagtaa cggggagagg gcacgggagg 2640 gctggatcag acgcttcctagagggacaga gacgaaggaa caatgcctag gcctcgggtg 2700 ggtgtgggac tggggactccccatcccccg caccccaccc acctcccgcg ggctccggat 2760 tatacgtgcg taagagtctggtgggatgga tttacggact tgaaaccgac ttctgctggc 2820 aggctttcac ctggatgggatatttgggtg gtgatgaggt ctttcccgag acacttttgg 2880 ttcagtcatt tgaaatgactttagagtagg gtgaggtggt gggaggctga tggagatatt 2940 gtgggggctt tagtccctccatggcaaagc agttcaggca aacaactcca tggttttccc 3000 tccaaattca aaaggccccgggtaacctgg aatccttcgt agtcggtttt gaagtggggc 3060 cttgggcgct gggggcatcaacatggccat ctgggcttgc ctgcccaggc cacacagagg 3120 ccccttgttg tgggtgaatggcaaagggaa gaggggactg gtgtggttca gaggccacag 3180 gctgggaaga gggatggcgggcgagtccaa ggaaactggc cgtgtcaccg tgcacctgcc 3240 acttcagccc cacgggtctataaaatgggc atgattatcg tggctacctc actggtcctg 3300 gcaattaagg aacaatgtgtgccaggcact ctgtaaacca catacttgcg agtgtcaagc 3360 tggtgacagg tggcgttcctgttgaagcac ctccctgagc tcacagcaac ccttgctgtc 3420 tctcctcttg ccctcagctcctgccagggc catccaggtg accgtgtcca acccctacca 3480 cgtggtgatc ctcttccagcctgtgaccct gccctgtacc taccagatga cctcgacccc 3540 cacgcaaccc atcgtcatctggaagtacaa gtctttctgc cgggaccgca tcgccgatgc 3600 cttctccccg gccagcgtcgacaaccagct caatgcccag ctggcagccg ggaacccagg 3660 ctacaacccc tacgtcgagtgccaggacag cgtgcgcacc gtcagggtcg tggccaccaa 3720 gcagggcaac gctgtgaccctgggagatta ctaccagggc cggaggatta ccatcaccgg 3780 aagtatgttg ggcagggcagggggatgagg ctgggcttgc ccgggtggtg ggactggcgt 3840 ccttgtgcgg gacctggagtccccatctga aagctcttga gtgccagtgt ctgaaaggac 3900 cattgaaggg agcaattctttttttttttt tttttgaaga tggagtcttg ctctggactc 3960 caggctggag tgcagtggtgcgatctcagc tcactgcaac ctccacctcc caggttcaag 4020 caattctctt gcctcagcctcccgagtagc tgggactcca ggtgcgtgcc accacgccca 4080 gttaattttt gtatttttagtagagatggg gtttcaccat gttggccagg ctggtctcaa 4140 actcctgacc tcaaatgatctgcccgcctt ggcctcgcaa agtgctgaga gacaccatac 4200 ccagcctaaa gggagcgattctattctact attcttcctt ctgctaatcc ttccattctt 4260 taatttaata acgaagattttttgagtacc tgtcatatac caggtgctgt tctgggccct 4320 gggaatacag ctgttaacaaaatcatcaaa ccacttccct cgtggagccc acattgcagt 4380 gagagagaca aacacgacacacactctcaa gtccttgaag ataaagaaaa ctgggtaacg 4440 gagagaagag gccagggtttgttctataat cattaataac acgagcagta agaagtaaaa 4500 tttatctaag taacaacttataaagggtct actgtgtgct aagctctcat ccaggttccc 4560 aaggattaac tcagaccacacagtaattga atagattcta tcattgtcat cttacagagg 4620 cccagagaga gaaagtgacttgcctagtgt catagctggt aacggggctg ggattctaac 4680 tcagccactt tgggtctagtggccaagctc ctaatccctt tgcttgccta gggtggtccg 4740 cagaggactc acagaggagatggcaggagt gaactgcagg ggcaagagag cttaatggag 4800 aaagcctgtg acatgccaggaactgcacac atattctccc attgagtcct ctcctctacc 4860 ctcctgacag ctgaggcacagagaggttac cttgttcaaa tgggtgcata ggaagtcaaa 4920 gtctggagct ggggtttgaacccaggcagc cctgagaacc ttgttctttt tttttnannc 4980 ggagtctcgc tctgtcgcccaggctggagt gcagtggcgg gatctcggct cactgcaagc 5040 tccgcctccc gggttcacgccattctcctg cctcagcctc ccaagtagct gggactacag 5100 gcgcccgcca ctacgcctggctaatttttt gtatttttag tagagacggg gtttcaccgt 5160 tttagccggg atggtctcgatctcctgacc tcgtgatccg cccgcctcgg cctcccaaag 5220 tgctgggatt acaggcgtgagccaccgcgc ccggcccctt gttcttaact gtaatgctgc 5280 ctcctgatag gatgtgcctgttgggactaa gtaaggggca gtcattcatt cattcatttg 5340 gtatttatca agcatcgactatgtgtcgtt ggtgctgggg atagaggtga ttgggatggc 5400 tgaagtttct gtcgtcaaggagatgacatt ctggtggagt nagactggca gtaaatnaag 5460 cagataaaga aagagtatgagaatttcaaa gtctgggcac ggtggctcac gtctgtaatc 5520 tcagcacttt gggaggccaaggtgggtgga tcacctgagg tcaggagttc cagaccagcc 5580 tggccaacat ggtgaaaccccgtctctact aaaaatacaa agattagcca ggcatggtgg 5640 cacatgcctg taatcccagctactcaggag gctgaggcat gagaatcgct tgaacccagg 5700 aggcagaggt tgcagtgagctgagatcgca ccactgtact gcagtntggg cgacagagtg 5760 agactctgtc tcaaaaaaaaaaaaaaaaaa aaagactccg tcaaggtata agaatgtcag 5820 agagtactaa gtgttgcaaagaaaataaca ccaggctggg tgcattggct catgcctgta 5880 aatttcagca ctttgggaggccaaggcagg aggatcactt gagcctagga gtttgagacc 5940 agcctggaca acaaaatgagaccccatgtc tacaaaaatt ttaaaaattt aaaaattagc 6000 tgggcatggt ggcatgtgcctgtggtcccg gctgctcagg aggctgaggt gggaggattg 6060 cttgggcttg agaggtcaaggcttcagtga gtcatgatcg tgccactgca ttccagcctg 6120 ggtgacagag tgagaccctgtcttgaaatg aaaagaaaat aggctgggcg cagtggctca 6180 cacctgtaat cccagcactttgggaggccg aggtgggtgg atcacctgag gtcaggagat 6240 cgagaccagc ctggccaacatggtgaaatc ccatctctac taaaaataca aaatttagcc 6300 gggcgtggtg gtgggcgcctgtaatcccag ctactcggga ggctgaggca ggagaatcgc 6360 ttgaacctgg gaggcgaaggttgcggtgcg ccaagattgc gccactgcac tctagcctgg 6420 gaaacagtga gactccgtcttaaaaaaaaa agaaaaaaga aaatagcact gggtgatgtg 6480 ctacatggaa tgacttgggctgtgaatatg atttgaggag ggcctgggcc tgggccttac 6540 agaacctaga aggcagagaggaaggggagg ggcagggtgc cagggatgaa ggctcacgta 6600 cctcatgtct tagtgtgtgttcactgtctt aaacaagaat ttaaagttgg gcatggggca 6660 gagcggggaa gggagcatccctttgcagac cccaagaagc caggaactgg agcacattct 6720 gctagaggat cgatgggaagcagggttcca ggggctgagc ctatgtcagt cctgtttcag 6780 aggaggcacc aggcttgcttgccctgaatt tctgtgggca gctcagccat gagcatccta 6840 ctgttattga ggtcacagggctgcttaggc cccctcctct ctaacccagg gattgtgcct 6900 gcctggacca ggcgtgactgctaagcttct gccaggacaa gccaaatact gagggtgctt 6960 cctctgctgg acgcaaaagtccaggatgac cccccaggct ctgtctcggg gaaggggccc 7020 tgcatgctcc aggggcctcacaggcctggg tctttcaaac cacccccacc tgggcctgtg 7080 tttgatcaag gccctgagtgtaaacatcca ttgtgtgtgt cctttcagga aatcccatag 7140 ccataggagc ttcctctgtttcagctttga ggatggggaa aagtggactc cccgtggtgt 7200 tcctagggtc acccactgtgctggggtttt tctgttgnnt gttgtttttt ttctgttgcc 7260 caggctggag tgcagtggtgcaatctcagc tcactgcaac ctctgcctcg caagttcaag 7320 tgattctccc gcctcagcctcctgagtagc tgggattaca ggtgcacacc accacacctg 7380 gctaattttt gtatctttttggtagagatg ggatttcgcc atgttggcca ggctggtctc 7440 aaactcctga cctcaggtgatctgcctgcc ttggcctccc aaagttctgg gattacagat 7500 gtgagccacc atgcccggcctatcctggtt tcaaaagtga aaatagtcct ggataaggta 7560 gaaggctgtc cactccaggcatccctccgg tccggtggct cattccctgc tttgtccttc 7620 catgctttgg gtgatggaccagcacctgga caggaggccc tgttccacct cctcgggctc 7680 cttggggtcc aagtgcccccacctccagct gcactgcagc agagagccca tgggacctct 7740 gaaatcatga aggtcacctttgcggtgtat aaagaaggaa ccagaggttg gagatgtgga 7800 ggaggcctgg ctgctgttcccactggagac ctggcatctt ctccccgacc taaaacaatg 7860 aaagcagtgc tcagcccggatgagatcacg gccagcccaa gaccaggaac agggtacgcc 7920 ctgcaggaag aaggtgtgcccagaccttag gatggatcaa aagaagccgg aaaactatat 7980 tttttgtgag ttttgaaaatgtcagacagg tcaaacaaaa cacagtgagg tccagcctcg 8040 gcctacaaga tgccagatttcaacccctgg cctatatgat ctgtttgcca tggcaggcgg 8100 ttcctgtcca cctcttttgtttatagcagg gaccagctct tgagctccag tgttgaagag 8160 gcacggtcag ggtctgatctgaagacactg gtggctcatg cctgtaatcc cagcacttca 8220 ggaggccgag gcaggaggattgcttgagga caggagctgg gagaccagcc tgggcaacac 8280 agtgagaccc agacactacaaaaaaataaa tttagcgggg catgatggca caccctgcta 8340 ctctggagat gggaagattgcttgagccta ggagttcgaa gctgcagtga cccatgatcg 8400 caccactgca ctccagcctgggcgaccaag ctaggccctc tcaaaaaaga tacaggtgga 8460 aaaatgatgg acgaagagggcattgtggca aacctgggga tttaggagaa cctagtttgg 8520 aattctatga ggattcaatgaaagaatgtg tgtagagggg cccagcacat agtaagagct 8580 caataaacgg tgggggctaggggtggtggc tcatgcctgt aatcccagca ctttgggagg 8640 ctgaggcagg tggatcacttgagccctgga gttcaagatc aacctggaca acaaagcaag 8700 atcccatctc aaaattaaaaaacaacacca acaacaaaaa aacagtggct tagatgcctg 8760 atcattaggg taagtcgtgtcctcaacccc ttcacatctg ctctgaaggt caccatatcc 8820 ggaagccttc cctggcctccttgtttaaaa tggcacagcc cccactccac gcctggcact 8880 ctctgctgtc cctgattcgttttctccata cagcttatct ttgtctggta tgtgacatag 8940 ttaacatttt atatttgtctttctttccta gttagaatct gaactctaga agggcaaggg 9000 caaggattta taactcaaagattccgggct taggcctctt ttatattctt gattttgagg 9060 ttaattaaga gctcaggcctagcgaggtgg ctcatgcctg gaatcccagc actttgggag 9120 gcccaggcgg gcagatcacttgaggtcagg agttccagac ctgcctggcc aacacagtga 9180 aaaacctgtc tctactaaaaatacaaaaat tagccagtta tgttggcagg cgcctataat 9240 cccagctact caagaggctgaggcaggaga atcgcttgaa cccaggaggc agaggctgca 9300 gtgagccaag atcgtgccactgcactccag cctgggcaac agagcgagac tccatctcaa 9360 aaaaaaaaaa aaaattaagagctcaaagag tttgttttca taggcagcag aatgagaaaa 9420 gtttacaaaa tagtttaaatgacaataaag tcattataga ttaacataaa taaaatacct 9480 tttatgaaaa aaataatcattttctgaaat cagacaaaac attgtgaatg agaaggtggc 9540 atggttttat ttttttgcaagtctccgaag cctggctgga tagaagagcc tggcttctca 9600 gagctgcttc agtctgttgtgatatctatt gtatgtcacg tagcctctgg aaaactccac 9660 agttagtatt gttgggaaaataactttgac ctcaggatct cctgaaaacg tcttggggaa 9720 ccccagggtc tagaggctgcagtttgagaa ctgttgctgt ggtatcccag gtgtctcaaa 9780 tactgcctag aacataggtggtactcagta attattgttg naaggatgaa tgaatgaatg 9840 aatgaatgaa tgaaagaaagaaatgtgtct ttgaatctag ccatgtgccc agaatgatga 9900 gacagatgac aaaagctaagggactttagc atgaggagag ggggttcgtt tccttttttt 9960 tctttttttt ttgagatggagtctcactct actgcccagg ctagagtgca gtggtgcaat 10020 ctcagctcac tgcaatctctgcctcctgag ttcaagcaat tctcctgcct cagcctccag 10080 ggtagctggg actacaggtgcgtgccacca tgcctagcta attttttaca tttttggtag 10140 agatggggtt ttaccatgttggccgggctg gtctggaact cctgacctca agtgatccac 10200 ctgcctcagc ctcccaaagtgttaggatta caggtgtgag ccaccatgtc cggccaagag 10260 ggtgttcatt tctgctccttgccaggtatt gtgtcaggca ctggggaccc agcagtggct 10320 gagacagaca gggctctgcctcacggagcc cacattttca ccaggcaaag gatggtcggc 10380 ccctaagctg ggagataagacttcagcagt tgggtggggg agccgtggga gaagcccagc 10440 ccacaggggg acagtgcaaatctagaacca aggcgatggc aggggtgagg ctggcacggt 10500 agctagagac cacgtcgtgccaagggcctt ggggaccatg ggactatggg accttaggga 10560 aggcgtctgg aatgctgtagccagacactg ttgcaaggag gatttttctg tagacatgag 10620 gccttcctta tgaagaaagcaagggttctt tcattcctgg gggtgccagg tgctgtggac 10680 tgcagcacgc gtggttgctgccgtcacaga gctgtcatgc aggagggcag cgcgtccttg 10740 ggaaggtggc aggcaggtcaggctaggagg aaagaggccg ggaagctgag ggcatttcct 10800 gcccgagatg cccaatgtagcctacttctg tccccagtgg cttaaggcag agttgcctgg 10860 taggtgccct ggtcccaccctggtgaaagg ctgaaggtat ttaattagtg cctgagaagc 10920 agagaggaaa caggatgtgccaaaacactt tgatggatgg tagagttaac aggctccttg 10980 cctgcagctg cttcagacaagagcgtcccc aagccctggg cctgacctgg aatgtgggga 11040 tggaagggga gggggaggaaccaaggcact gggagggtaa gtctctctct cccacataga 11100 cacacccact ccttatgggtgcctgggcat ctcctggtac ctagaatctg gcctgtttat 11160 ctccacaccc atccctggggtctacactag gccctgtggg tggcagttca catcagggga 11220 gttctgactt tggctctgagaggtggttca gagatggctg taagttgaga agcacagact 11280 gctgggtgtg gtggttcacgcctgtaatcc cagcactttg ggaggctgag gtgggggtgg 11340 atcacctgag gtctggagttcaaaaccaac ttggtcaaca tggcgaaact ccatctctac 11400 taaaaatgca aaaattagccaggtgtggtg gcaggtgcct ataatcccag ctacatggga 11460 ggctgaggca ggagaatcgcttgaatctgg gaggcgaaga ttgtagtgag ccgagattag 11520 ttcgcaccat tgcatgccagcctgggcaac aagagtgaaa ctccgattca aacaaacaaa 11580 aaaaaaaagc tgggcatggtggagtgcctg tagtcctaac tactcaggtg ggaggattgc 11640 ttgagtccag gaggttgaagttgcagtggg ctataattac accactgcac tccagccagg 11700 gccacagagt gagaccctgtctctaaagaa agaaaaaaaa aaacaacctc aggctccgag 11760 ggcaccatta ctgctctacactgaagagct gtgcagcttt tccagacccg aaatgtcatc 11820 cacaaaacag aagtgataatggtcctgcct cacagacttc ttgcagtagt ccaggtgttt 11880 agaacggggt gtaaaaggccgtgtgccctt ggtaggaatc ttngcatatg catttgatca 11940 tctgcagcct gcccagcccactgcttgccc cctcctgggt gtgctgggaa ggggtctttg 12000 gccctccagg ggttaggtgccccagcctcc aaggtgccct cacgcctttt catcccgact 12060 cagatgctga cctgacctttgaccagacgg cgtgggggga cagtggtgtg tattactgct 12120 ccgtggtctc agcccaggacctccagggga acaatgaggc ctacgcagag ctcatcgtcc 12180 ttggtgagtg ggcctgggaagggggaggca tggcccttcc ttttgtccgc ttctgttctg 12240 tctgccctcc cctgtgtccgccctctgccc tccagcttac cctctgggct ctgtcgcctg 12300 ctctgctctc ccccaggctctgccagtcac ttaggctccc ctgtgccctg caccccaggc 12360 agggaccact ggcccacagtgcctccaatc acccaagcca aactaagaga agagtggaga 12420 caattggaga ctctgccttttcaaagtctc atttttaaaa aaaatccaga cttggggtcc 12480 gggtgcggta gttcatgcctgtaatcccag cactttggga ggccgaggcg ggtggatcac 12540 ttgaggccag gagttcgagactagcctggc caacgtggca aaatcccgtc tctataaaaa 12600 atataaaagc caggcgtggtggtgcacatg cctgtaatcc cagttactca gaaggctgag 12660 gcatgaggat tgcttgaacctgggaggcag aggatgcagt aagccaagat caagccactg 12720 cactccagcc tgggcgacagagtgagactc tgtccaaaaa aaaaaaaaat ccagacgtgg 12780 tcagagtcca tgggcagtgaatgaggacag ttgatggtgt gcaaaatcga cccacctctt 12840 gctacatccc caaggcctcatctcacccga gtccctcgcc aaagcacagc ggttttgccg 12900 tgtgccctgc tgggatggcgctgcatggca cacacactgt gtaagtttga gtgcagctga 12960 aacgaagccg attccagacacccaggggca gggcggggtg tccgtgtggc tgggaggcct 13020 ccttgtgtta gggggatgttgccatcggcc aggtgccctg ctgtaagcca acacatggag 13080 tcttgtatga catgtgctctgcatgagtga tgccgctggg ctgtacactg ccatcttcac 13140 atgtgtgaat gagcacgtgactggggggta cttgggctgc aagacagagt tcatgtgtgg 13200 gggatggaac acgtgcaccagtgacccagg aacctctgcc tgttcttcgg taaaatgcac 13260 catttgcatc agcagttcccaaaattagtc tccaggtcta tttacactct aaaacattat 13320 cgagggtctc caagagcttttgtttgtttc tgtgggtttt atgtctatct gttgcttaac 13380 atattaggaa ttaaaatggggagattttcc tttttttttt tttttttttg agatggagtc 13440 tcgttctgtc gcccaggctggagtgcagtg gctcgatctc ggctcactgc aagcttcacc 13500 tcctgggttc acgccattctcctgcctcag cctcccaagt agctgggact acaggcaccc 13560 gccaccacac ccggctaattttttttgtat ttttagtaga gactgggttt caccatgtta 13620 gccaggatgg tctcgatctcctgacctcgt gatccaccca cctgggcctc ccaaagtgct 13680 gggattacag gcatgagccactgcccggcc ttaaaatggg gagatttttc aagcccaaga 13740 tacacaagga agactgggcaacatggcaag accctgactc tacaaaaaat tttaaaatta 13800 accaggcatg gtggcatgcacctgtgagcc cagcttcttg ggaggctgag gcaggagtat 13860 cgcttgcacc caggaggtcaaggctgcagt gagccatgac tatgctactg cactctagca 13920 tgagtgacag agaccctggctcaagaaann canacaaaca cacacacaca cacacacacg 13980 catatagtcc attaggcatcagggcgatga tggcatcagg gagcctggga aactctactg 14040 gacattcatg ggagaacaagtgaaaaaggc aaataacatc ttagtgttat tctaaaattt 14100 cttcttttgg ccttgtggacaggaccacgc tttgagagct gtgactgaca tgcctctgtc 14160 ctgttgcgag ggcctatagtgccaagtgca tgagctctgg ggagggcttc gtgggtgcag 14220 agctgggcct gtggaggcccctcagacaca acactggtgg ggctcagagc tccaggggca 14280 ctcgagggaa gacaagaaccggctctgaga tgcgtgaatg tgacagtgca tgagtagaga 14340 tggagacctt gtgggtcccagaaccaggac tgcatatgac tttcatatgt gggtattttt 14400 gccttcatgg gtcccttcctgttttaaaaa aaatgtgtga ttatgttgtc acaaagagtt 14460 tattcctgta tattgtgttaatttgtgttc agatttgtaa agtaaaatta aaccatttca 14520 gccaggtgtg gtgacacatgcctgtagccc tagctactta ccccagaggc tgaggtggga 14580 ggatcgcctg agcccacgaggttgaagctg cagtgagcca tgatcacacc cctgcactcc 14640 agactgggcg acagagctgagatcctattt cgtgggccct aggtccctgt gcctgctgga 14700 acaggacatc cctatcaccgtggttggagc cctttggggt gctaagacct atgaatgagg 14760 gaaacttagg gtgcccaagctgaggtagag ccctcagaac cccctgggat ttgtattgga 14820 gccctcgtgg cataacacaggtggattatg caatgggagt ttcttaccta taagcaccca 14880 catgtgggcg ggtggagggtaggagccatg cgctagggct tcagccccca gccccttccc 14940 gcttcagggc acaccttgcacttggccagc ctggagctgg gctttcgggg gtggcacagc 15000 ctgggctggc tctggccagcataatctgtt tctcttttgt ccctccaggg aggacctcag 15060 gggtggctga gctcttacctggttttcagg cggggcccat agaaggtacg gggggtggat 15120 cctgagttgg gcttctcgggagctcccata catcacctac tgcttctgac tctagttagt 15180 atccccttcc ccactaaaccctgctcactg tggacccctc actaacctgg cctgactgtg 15240 gctctgaggc atctagtggtctggcgctgg gcctaggcta ggctgggctg aggagagcct 15300 ggggtgcagg ccagggctctgtgactggca cctgcggtgc tcttgagggt gtggcgtctg 15360 ggcagctggc tctctctttggtctgggggc tgcagtctgt ctccctctgt gcaggctgcc 15420 tcgttttctg ccttgtgttttttgcacctg ggggagggcc gtaactgggg aatggccggg 15480 atggtagaat ggggagtgtgctgtgcccag cctctggcac aaaaaatcca gccagggctg 15540 caggttcctt ggtgagctttgcaaatcgtc cccgacctca gtgctggctc cgcaccatgt 15600 acccctgctg tgccgttagccctgttccct cccaggcctc cgggctcagg gcctgttgtc 15660 tttctgcaga ctggctcttcgtggttgtgg tatgcctggc tgccttcctc atcttcctcc 15720 tcctgggcat ctgctggtgccagtgctgcc cgcacacttg ctgctgctac gtcaggtgcc 15780 cctgctgccc agacaagtgctgctgccccg aggcccgtaa gtgtcccgct catggccacc 15840 ctggtttggg caacatcctgcatccaaggg aaggaggtgg ccatccacct gcccccagga 15900 cagtggcgtt ggtctggagggtgtgaattt agccagtggg gagaaagtag gctgaggagg 15960 gtctgctgtt tagattgtcgtttacttcct ccaactttta gtttattttt atttatgttg 16020 ttcttttctt ttgtaagtataatccataca catggtaaaa atgtccaaca gtacaagata 16080 ctagtcacat ggaagtaaagccctctaaaa aaaccaaatc ttggctaggc gcagtgatta 16140 cgcctgtaat cccagcactttgggaggcca agacgagtgg atcacttgag gtcaggagtt 16200 ccagatcagc ctggccaacatggtaaaacc cagttctcta ctaaaaatac aaaaattagc 16260 tgggcatggt ggtgatcgcctgtaatccca gctactcagg agactgaggc atgagaatcg 16320 cttaaaccca agaagtggaggttgcagtga gctgagatca cgccactgca ctccagcctg 16380 ggcgacagag tgagactctgtctcaaaaaa aaaagaaaaa aaaatgttaa gtgaaaaagt 16440 taagaaacca aacaaggtttacaacactac atgatttaag caaaaaaaat tttttttgtt 16500 ttagagaaag ggtctcattctgtcatccag gcagtgcagt gcgatcatag ctctctgcag 16560 cctcaaactc ccgggttcaagcagtcctcc cgcctcagcc tctggagcag ctgggactgt 16620 aggcacacac caccatgcccagctaatttt ttgatttttg ttttttgtag agacggggtc 16680 tcagtatgtt gcccagcctgatctcaaact cctggcctca ggtgatcctc cgaagtcagc 16740 ctccccaaag tgctgggattacaggcatgt gccaccatgc tggccaattt ttaaaaattt 16800 tctgtagaga cagggtcttgctatgttgcc caggctggtc ttgaactctt gacctcaagt 16860 gatcctgcct caggctcccaaagtgatggg attacaggca tgaactacca cacctggcct 16920 taaacttaag caaatttttttttttttttg gagacagttt cactctgtcg cccaggctgg 16980 agtaaagtgg cgtgatctctgctcactgca acctccgccc cccgggttta agctattctc 17040 ctgcctcagc ctcccgagtagctgggatat aggcgcctgc caccacgcct gactaatttt 17100 tgtattttta gtagagacggggttttgcca tgttggccag gctggtctcg aactcctgac 17160 ctcaggcaat ccgctcccccgcacccctac cttggcctcc caaagtgtta ggactacagg 17220 tgtgagccac catgcctggccaaatttaag caaatgtttg aaaacacata cccacaggaa 17280 tgctgcacat tttacccagctactatgtct agggtcgtat ctagcacacc agcatggcta 17340 ctgtggagag ctgggactggatgtgagatg agagctaaag gggaagtaag caaaccaagc 17400 aggggaaggt aagagaagacagaagacaga gagagaggga cctaactcta tgagaggagt 17460 cagacatgtg caattgaaaaagacttgctc ctgtctctct tctgtgaatg tttgtgaata 17520 tcccaacggg acactttcacagaggagctg attgacgtgg tcacagccat cagccttggg 17580 acaccagacc acagtgtgtacactaagtgg cactgatgga cacttcagca tccctctagc 17640 tgctgtcccg tttcccctcctcggggacca cagctgttgc cagtccttgg tttccttcag 17700 gagggtgtct gggtagaccagcctgtgtgc acacagtcca agatacatga acagtgaagt 17760 gccaggcaat ccttgcaagcatgggcaggt ggagagctga ggcctgcttg acaccttcct 17820 gctcagaagc ccagtgagcagtttccctcc ctagggctca gtgtcatccc ctataaaatg 17880 gggcttatgg cagagctcaccacactgggt gcatctgggg atttggcgag ctcatgtgca 17940 caccattgag catggggcccaacctatata aaatattcta cgtctgtcag ctgctgggca 18000 ctgccactat cagcctcagtagtgactgag ggacagggca ccagtcagag ccctggtgca 18060 cacagagtga ccccagagaagcagccttcc ctctctgagt cctgtttcct tctgttaggt 18120 cctgacttca tgggttgttgttagcattaa ggaagtcgct ggctaatttt atagtcattg 18180 aagtcagtgg tgtgcaacctggttcctcaa aggatcactt ccctgaaaaa attccactgc 18240 tccctggagg cttatgcaggccatcccatc ccctccctct tgttgtgttc agctgacagc 18300 tttttgctca gtgagtaagtgttaggtcca tttcacagat gggctgcaac caagtttgca 18360 gtgaacccac taagaccagagctagggcca ggactaaatg ctggtcccaa tgccacattc 18420 ccctgtcccc acaccacatttcctccatcc ggagaccctg ttaccccaac ccagggcccc 18480 attaactccc tggcagaggccctgttacat ctgctgctgc cacagcctcc gcccaccctt 18540 caggaggcag caggtcccactgctgatgat aaagttgcag gctgcctgag ctaatgaagg 18600 ggcttcctct aggctgtgcacttagtcttc tgcttccaaa ccaaatcaga ggtgaggcac 18660 cctctctggg cccatctctctcctccattt tcctgttggg gtcccaggga ggaagccact 18720 tgcctagggc ccaggaattttgcaagcctc ttgccctagg gaggaaggaa gggaggagga 18780 tcttaccttg aactgtcaagcctagagcct ggtggggcag gcagaaatgg gtgcagtcca 18840 tgagttagaa acactagaggagacactttg ctgcttggnc cggggcaggc aagnttaatt 18900 cccgaggctc ctgccactgcatctcaatct ggaaggtgac caggtggggc aggacccacg 18960 tctcccagat gactcattttttctagaaca ggggcttggc tgccaaagag gatacttgat 19020 ttcggcttgt ggggacagtggtggacccag catctgggct ttatataaag ggcagctttg 19080 ttgccctgta aacacacagaccatgggtgg ccacttcttc cagtaagtta gctggggagt 19140 tggaagttta ggtaaaaccttttgattgac aaatgttggc gaattaccat gctgttaaat 19200 gaaacattgt tctgccaccctggggctgtg ggtgcctgcg tgcaccctct gaaaaatcac 19260 acaggaagtg gggtggggtctctgtgaagc tggtgtcccc cagcctcagg gatgctgcag 19320 aaatggaatg aggaccaacagggactcaga tgtccaagga agctctacag cggagaggac 19380 ggcttgggaa ggaggtccaggcccaggtcc ctccggaacc caatgggtat ggggcagcct 19440 ggctcctgcc tcatcccccttctcctgttg attatgtcct cacagtgtat gccgccggca 19500 aagcagccac ctcaggtgttcccagcattt atgcccccag cacctatgcc cacctgtctc 19560 ccgccaagac cccacccccaccagctatga ttcccatggg ccctgcctac aacgggtacc 19620 ctggaggata ccctggagacgttgacagga gtagctcagg tgaggccggg ggaagcagga 19680 acagctggtg ggagtgtgctgggcatctgg acactgaggg gcaggggctg gaaggaagag 19740 tgtcttggga gccgaggaggggctctgctc ctggtgcgcg gccactgaca gccactctcc 19800 cccagctggt ggccaaggctcctatgtacc cctgcttcgg gacacggaca gcagtgtggc 19860 ctctggtgag aatccatcgtcccgaagttg gatgtgcctg taagggagag gggtgggcca 19920 ggatccatcc tcccaaaccgaccaccaccc ccctgtccct agaagtccgc agtggctaca 19980 ggattcaggc cagccagcaggacgactcca tgcgggtcct gtactacatg gagaaggagc 20040 tggccaactt cgacccttctcgacctggcc cccccagtgg ccgtgtggag cggggtaagc 20100 aggagccttg gggtctgagggcttttaagg tgggggggtg aaacatgtct ccctgatacc 20160 tgccgcaggg actcttggtgcaaaccctgg accccgggct cctccagcag tcagtgacac 20220 cccccttccc tgcagccatgagtgaagtca cctccctcca cgaggacgac tggcgatctc 20280 ggccttcccg gggccctgccctcaccccga tccgggatga ggagtggggt ggccactccc 20340 cccggagtcc caggggatgggaccaggagc ccgccaggga gcaggcaggc gggggctggc 20400 gggccaggcg gccccgggcccgctccgtgg acgccctgga cgacctcacc ccgccgagca 20460 ccgccgagtc agggagcaggtctcccacga gtaatggtgg gaggagaagc cgggcctaca 20520 tgcccccgcg gagccgcagccgggacgacc tctatgacca agacgactcg agggacttcc 20580 cacgctcccg ggacccccactacgacgact tcaggtctcg ggagcgccct cctgccgacc 20640 ccaggtccca ccaccaccgtacccgggacc ctcgggacaa cggctccagg tccggggacc 20700 tcccctatga tgggcggctactggaggagg ctgtgaggaa gaaggggtcg gaggagagga 20760 ggagacccca caaggaggaggaggaagagg cctactaccc gcccgcgccg cccccgtact 20820 cggagaccga ctcgcaggcgtcccgagagc gcaggctcaa gaaggtgagg gccgccctcc 20880 ctggcgtcca gaccgtccctgggcccccag ccggtccccg cggctcatac ccttctttct 20940 ttctcccttg cagaacttggccctgagtcg ggaaagttta gtcgtctgat ctgacgtttt 21000 ctacgtagct tttgtatttttttttttaat ttgaaggaac actgatgaag ccctgccata 21060 cccctcccga gtctaataaaacgtataatc acaagctctg gagagaacca tttgttcggc 21120 cgcgcggggc gggggaccggggctgctccc gtatgcgtct gtaaagcgcc gcgtcccggg 21180 ggcaccggag tccggggccgggaggaagag acccagcctg gcccggcccg cgcccgcgcc 21240 gccggccgga gaacgtgccccgcgcagcca ccgcccgcct gcgtgcgcgc cccggccccg 21300 cccaggcgtg cgcatgcgccccggccctcc gccttcgcgc accgcaggct ggccgccggg 21360 agcgcgcgcg cgctcctctccccttccagc ccatcccccc cagcccccca ccgacctact 21420 ttactgtctc caaactcgggcagcccacct ggcccccgac gaccccagcc cctgctccgg 21480 gtaccccgac gttccatccagacccgcgtt tcaccagggc ggcgcgcggc gacctcgcgc 21540 cccgcggagc cccgggctcgcgcgcgcccg cccgcccccg gagacagaca gcgcgcgcgc 21600 tcccgggccg cctccccccagcgcgcgtcc gccccgggct cgcgccgccg ccgccgccgc 21660 cgccgcgcgc gcgcagctcaagtaaaggag gaaaaaaaaa agggggaaaa atagaaagcg 21720 g 21721

We claim:
 1. A purified or recombinant Lipolysis Stimulated Receptor,wherein said receptor comprises a polypeptide comprising at least 10 to15 consecutive amino acids of SEQ ID NO:
 8. 2. The Lipolysis StimulatedReceptor of claim 1, wherein said polypeptide a) comprises the aminoacid sequence of SEQ ID NO:8; or b) consists of the amino acid sequenceof SEQ ID NO:8.
 3. The purified or recombinant Lipolysis StimulatedReceptor of claim 1, wherein said receptor comprises a biologicallyactive polypeptide comprising an amino acid sequence selected from thegroup consisting of: a) an amino acid sequence spanning amino acids 76to 94 of SEQ ID NO:8; b) an amino acid sequence spanning amino acids 76to 160 of SEQ ID NO:8; c) an amino acid sequence spanning amino acids 76to 237 of SEQ ID NO:8; d) an amino acid sequence spanning amino acids157 to 249 of SEQ ID NO:8; e) an amino acid sequence spanning aminoacids 236 to 530 of SEQ ID NO:8; f) an amino acid sequence spanningamino acids 236 to 613 of SEQ ID NO:8; and g) an amino acid sequencespanning amino acids 76 to 613 of SEQ ID NO:8.
 4. A recombinant cellexpressing the recombinant Lipolysis Stimulated Receptor of claim
 1. 5.A recombinant cell expressing the recombinant Lipolysis StimulatedReceptor of claim
 3. 6. A method for selecting a compound useful forenhancing lipoprotein uptake in cells comprising the steps: a)contacting the recombinant cell of claim 4 with a candidate compound inthe presence of a lipoprotein; and b) determining whether the amount ofinternalized lipoprotein is greater in the presence of said compoundthan in the absence of said compound, wherein a determination that saidamount of internalized lipoprotein is greater in the presence of saidcompound indicates that said compound is useful for enhancinglipoprotein uptake in cells.
 7. The method of claim 6, wherein saidcandidate compound is a small molecule.
 8. A method for selecting acompound useful for enhancing lipoprotein uptake in cells comprising thesteps: a) contacting the recombinant cell of claim 5 with a candidatecompound in the presence of a lipoprotein; and b) determining whetherthe amount of internalized lipoprotein is greater in the presence ofsaid compound than in the absence of said compound, wherein adetermination that said amount of internalized lipoprotein is greater inthe presence of said compound indicates that said compound is useful forenhancing lipoprotein uptake in cells.
 9. The method of claim 8, whereinsaid candidate compound is a small molecule.
 10. A recombinantpolypeptide comprising the amino acid sequence of SEQ ID NO:8.
 11. Thepolypeptide of claim 10, wherein said polypeptide consists of SEQ IDNO:8.
 12. An isolated or recombinant biologically active polypeptidecomprising an amino acid sequence selected from the group consisting of:a) an amino acid sequence spanning amino acids 76 to 94 of SEQ ID NO:8;b) an amino acid sequence spanning amino acids 76 to 160 of SEQ ID NO:8;c) an amino acid sequence spanning amino acids 76 to 237 of SEQ ID NO:8;d) an amino acid sequence spanning amino acids 157 to 249 of SEQ IDNO:8; e) an amino acid sequence spanning amino acids 236 to 530 of SEQID NO:8; f) an amino acid sequence spanning amino acids 236 to 613 ofSEQ ID NO: 12; and g) an amino acid sequence spanning amino acids 76 to613 of SEQ ID NO:8.
 13. A recombinant cell expressing the recombinantpolypeptide of claim
 10. 14. A recombinant cell expressing therecombinant polypeptide of claim
 12. 15. A method for selecting acompound useful for enhancing lipoprotein uptake in cells, comprisingthe steps of: a) contacting the recombinant cell of claim 13 with acandidate compound in the presence of a lipoprotein; and b) determiningwhether the amount of internalized lipoprotein is greater in thepresence of said compound than in the absence of said compound wherein adetermination that said amount of internalized lipoprotein is greater inthe presence of said compound.
 16. The method of claim 15, wherein saidcandidate compound is a small molecule.
 17. A method for selecting acompound useful for enhancing lipoprotein uptake in cells, comprisingthe steps of: a) contacting the recombinant cell of claim 14 with acandidate compound in the presence of a lipoprotein; and b) determiningwhether the amount of internalized lipoprotein is greater in thepresence of said compound than in the absence of said compound wherein adetermination that said amount of internalized lipoprotein is greater inthe presence of said compound.
 18. The method of claim 17, wherein saidcandidate compound is a small molecule.
 19. The polypeptide of claim 10,wherein said polypeptide combines with one or more heterologouspolypeptides to form an LSR receptor complex, and wherein said complexcomprises an α subunit or an α′ subunit, and at least one β subunit. 20.The polypeptide of claim 19, wherein said complex comprises three βsubunits.
 21. The polypeptide of claim 19, wherein said polypeptide isfrom a human, and wherein said polypeptide has a molecular weight of 64kD.
 22. The polypeptide of claim 19, wherein said polypeptide isexpressed in hepatic cells.
 23. The polypeptide of claim 19, whereinsaid complex has a biological activity selected from the groupconsisting of lipoprotein binding, lipoprotein internalization, andlipoprotein degradation.
 24. The polypeptide of claim 19, wherein saidcomplex has a biological activity that is selected from the groupconsisting of leptin binding, leptin internalization, and leptindegradation.
 25. The polypeptide of claim 12, wherein said polypeptidecombines with one or more heterologous polypeptides to form an LSRreceptor complex, and wherein said complex comprises an α subunit or anα′ subunit, and at least one β subunit.
 26. The polypeptide of claim 25,wherein said complex comprises three β subunits.
 27. The polypeptide ofclaim 25, wherein said polypeptide is from a human, and wherein saidpolypeptide has a molecular weight of 64 kD.
 28. The polypeptide ofclaim 25, wherein said polypeptide is expressed in hepatic cells. 29.The polypeptide of claim 25, wherein said complex has a biologicalactivity selected from the group consisting of lipoprotein binding,lipoprotein internalization, and lipoprotein degradation.
 30. Thepolypeptide of claim 25, wherein said complex has a biological activitythat is selected from the group consisting of leptin binding, leptininternalization, and leptin degradation.
 31. The polypeptide of claim12, wherein said polypeptide is recombinant.
 32. A compositioncomprising the polypeptide of claim
 10. 33. A composition comprising thepolypeptide of claim
 12. 34. The composition of claim 32, furthercomprising a physiologically acceptable carrier.
 35. The composition ofclaim 33, further comprising a physiologically acceptable carrier.
 36. Amethod of making the polypeptide of claim 10 comprising the steps of: a)obtaining a cell capable of expressing said polypeptide; b) growing saidcells under conditions suitable to produce said polypeptide; and c)isolating said polypeptide produced by said cell.
 37. The method ofclaim 36, wherein said cell is prokaryotic.
 38. The method of claim 36,wherein said cell is eukaryotic.
 39. The method of claim 36, whereinsaid cell is recombinant for polynucleotide encoding said polypeptide.40. The method of claim 36, further comprising purifying saidpolypeptide produced by said cell.
 41. A method of making thepolypeptide of claim 12 comprising the steps of: a) obtaining a cellcapable of expressing said polypeptide; b) growing said cells underconditions suitable to produce said polypeptide; and c) isolating saidpolypeptide produced by said cell.
 42. The method of claim 41, whereinsaid cell is prokaryotic.
 43. The method of claim 41, wherein said cellis eukaryotic.
 44. The method of claim 41, wherein said cell isrecombinant for polynucleotide encoding said polypeptide.
 45. The methodof claim 41, further comprising purifying said polypeptide produced bysaid cell.